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查看更多产品信息 Novex™ Tricine Welcome Pack, 10-20% - FAQs (EC6625A, EC6625B, EC6625C)
193 个常见问题解答
以下是一些可能的原因和解决方案：
- 凝胶上的分子量标准品的量不足： 将适当体积的分子量标准品上样到凝胶上。以下是我们的建议：
•小型凝胶：每孔5μL（厚度0.75-1.0 mm）或每孔10μL（厚度1.5 mm）
•大凝胶：每孔10μL（厚度0.75-1.0 mm）或每孔20μL（厚度1.5 mm）
- 转印不完全或不理想： 优化转印条件
以下是一些可能的原因和解决方案：
- 样品被煮沸： 丢弃煮沸的等分样品。
- 使用的分子量标准品体积过大：使用前加入较少的体积或稀释蛋白质上样缓冲液中的分子量标准品。
褪色最可能是由于封闭/洗涤液中的洗涤剂将一些蛋白质从膜上洗脱造成的。染料本身不会从蛋白质上洗脱，因为它们是共价结合的。我们已经发现较小孔径的膜可以在封闭和洗涤过程中更好地保留蛋白质，因此，为了获得绝佳的分辨率和蛋白质保留率，推荐使用0.2 µm代替0.45 µm的膜。转印后，可以用铅笔在膜上圈出预染条带，从而在封闭和处理后仍可辨认条带位置。
•降低电压、电流或缩短转印时间
•确保转膜缓冲液的甲醇浓度合适；可使用浓度为10–20%的甲醇，从而去除SDS-蛋白质复合物中的SDS，并促进蛋白质与膜的结合。
•确保转膜缓冲液的SDS浓度合适（若加入了SDS），SDS浓度不要超过0.02–0.04%。过多的SDS会阻碍蛋白质与膜的结合。过多的SDS会阻碍蛋白质与膜的结合。
•检查膜的孔径和靶标蛋白质的大小。小于10kDa的蛋白质很容易穿过0.45μm孔径的膜。如果您的目标蛋白质小于10 kDa，那么最好使用0.2μm孔径的膜。
•增加电压、电流或转印时间
•凝胶和SDS-蛋白质复合物中的SDS会促进蛋白质从凝胶中洗脱，但抑制蛋白质与膜的结合。这种抑制作用在硝化纤维素膜上的强度大于PVDF膜。对于难以从凝胶中洗脱的蛋白质，如大分子量蛋白质，可在转膜缓冲液中加入少量SDS以改善转印效果。我们建议在组装三明治前将凝胶置于含0.02–0.04% SDS的2x转膜缓冲液（无甲醇）中预平衡10分钟，然后使用含10%甲醇和0.01% SDS的1X转膜缓冲液进行转印。
•甲醇可去除SDS-蛋白质复合物中的SDS，促进蛋白质与膜的结合，但对凝胶本身有一些不良影响，会降低转印效率。甲醇可能导致孔径减小、某些蛋白质发生沉淀以及一些碱性蛋白质带正电荷或变为中性。应确保转膜缓冲液的甲醇浓度不高于10–20%，并使用高质量的分析级甲醇。
预染标准品具有与每种蛋白质共价结合的染料，这将导致标准品在不同的缓冲系统（即不同的凝胶）中迁移率不同。因此，使用预染标准品进行分子量估算将仅得出蛋白质的表观分子量。预染标准品可用于分子量估算、确认凝胶迁移和估算转膜效率，但对于需要精确估算分子量的应用，应使用非预染标准品。
•上样时，请注意确保相邻样品泳道没有交叉污染。
•确保每个泳道上标准品的量都是正确的。蛋白质上样过多会导致产生额外的条带，这个问题在使用银染凝胶时尤为突出。
•标准品储存不当或反复冻融会导致蛋白质降解。
建议如下：
•确保每个泳道上标准品的量都是正确的。蛋白质上样过多会导致模糊，这个问题在使用银染凝胶时尤为突出。
•条带在低百分比凝胶中不能很好地分辨。尝试使用更高百分比的凝胶。
•如果在转膜/检测后条带看起来不明显和模糊，可能是由于抗体浓度过高。遵循制造商建议的稀释度或通过斑点印迹确定最适抗体浓度。
建议如下：
•检查使用的凝胶类型/凝胶百分比。可能会由于凝胶类型和/或百分比的不同而不能看到所有条带。例如，蛋白质标准品的最小条带可能不能在非常低百分比的凝胶上分辨，而较高分子量条带可能不能在高百分比凝胶上分辨。
•检查蛋白质分子量标准品的有效日期。由于蛋白质降解，过期批次可能导致条带褪色或缺失。
•检查蛋白质分子量标准品的储存条件。不适当的储存条件会损害标准品中蛋白质的稳定性。
•确保蛋白质分子量标准品在上样到凝胶上之前未加热/煮沸。我们的蛋白质分子量标准品可直接上样，我们不建议将其加热/煮沸，因为这可能会导致标准品中的蛋白质降解。
小型转印模块专为小型凝胶电泳槽设计。该模块也适用于Bolt小型凝胶电泳槽（2014年12月31日起停产），但不适用于XCell SureLock Mini Cell或其他供应商生产的电泳槽。
由于通用的电极设计，小型转印模块可装在小型凝胶电泳槽的任意一侧。
是的，我们提供小型转印模块（货号B1000），适用于小型凝胶电泳槽。该转印模块也可用于Bolt小型凝胶电泳槽（2014年12月31日起停产）。请注意，Bolt小型转印模块（2014年12月31日起停产）对Bolt小型凝胶电泳槽和小型凝胶电泳槽均适用。
Bolt Bis-Tris Plus凝胶也可使用XCell SureLock Mini Cell、iBlot干转系统或Invitrogen半干转仪进行转印。
氯丁醇可用作NuPAGE转膜缓冲液的防腐剂，但对于蛋白质的有效转印并不是必要的。若配制缓冲液时未加入氯丁醇，则应注意缓冲液不能长期稳定保存。建议在配制后2周内用完。
我们不建议使用碳酸盐或CAPS转膜缓冲液进行NuPAGE凝胶转印，因为这会降低转印效率。此外，这些缓冲液的高pH环境（>pH 9）会使NuPAGE抗氧化剂丧失功能。
为提高NuPAGE凝胶上大分子蛋白[高分子量蛋白]的转印效率，我们建议在安装“三明治”前，将凝胶置于含有0.02–0.04% SDS的2x NuPAGE转膜缓冲液（无甲醇）中预平衡10分钟，然后使用含甲醇和0.01%SDS的1XNuPAGE转膜缓冲液进行转印。
我们建议在将凝胶盒放置在电泳槽中之前，使用记号笔在凝胶盒上标记出孔的底部。
以下是可能原因和解决方案：
- 凝胶盒底部留有胶带。应从凝胶盒底部去除胶带。
- 与电源连接不良。使用电压表检测所有连接处的电导率。
- 缓冲液不足。应确保电泳槽中的缓冲液足以浸没凝胶的上样孔。
以下是可能原因和解决方案：
- 使用了浓度过高或错误的缓冲液。应检查缓冲液配方；必要时，稀释或重新配制。
- 电流设置过高。将电流降低至推荐的电泳条件（见使用手册第8页）。
以下是可能原因和解决方案：
- 缓冲液稀释过度。检查缓冲液配方；必要时，重新配制。
- 缓冲液槽泄漏。确保凝胶盒夹安置稳固，垫片在原位，并且凝胶盒夹已锁定。
- 电流设置过低。设置正确的电流。
以下是我们可提供的小型凝胶电泳槽替换部件：
替换部件 - 货号
凝胶电泳槽 - B4478641
小型凝胶电泳槽盖 - A25944
小型凝胶电泳槽基座 - A25950
凝胶盒夹，左 - A25946
凝胶盒夹，右 - A25945
凝胶盒夹凸轮手柄装置 - A26732
我们建议：
•电泳完毕后，妥善处理电泳缓冲液。用水冲洗电泳槽以除去残留的缓冲液。
•用蘸过水的柔软非摩擦性无尘布清洁小型凝胶电泳槽的表面。
•不要使用强力洗涤剂或溶剂清洗装置。
小型凝胶电泳槽不兼容氯化烃（如氯仿）、芳香烃（如甲苯和苯）或丙酮。
在使用转接板的情况下，Precise Tris-HEPES凝胶可兼容XCell SureLock Mini Cell或小型凝胶电泳槽。
注意：使用XCell SureLock Mini Cell或小型凝胶电泳槽时，1块凝胶需要2个转接板，2块凝胶需要1个转接板。
我们的传统Bolt Bis-Tris Plus小型凝胶（货号BGxxxxxBOX，2014年12月31日起停产）只能使用Bolt小型凝胶电泳槽（2014年12月31日起停产，库存售尽后将停止供应）进行电泳。
我们的新型Bolt Bis-Tris Plus小型凝胶（货号NWxxxxxBOX）以及Invitrogen小型凝胶和NuPAGE小型凝胶，可使用小型凝胶电泳槽或XCell SureLock Mini-Cell。若这些凝胶使用Bolt小型凝胶电泳槽（2014年12月31日起停产）进行电泳，则有必要升级电泳槽，可将黑色10.5 cm凝胶盒夹凸轮手柄更换为灰色10 cm凝胶盒夹凸轮手柄（货号A26732，凝胶盒夹凸轮手柄装置）。点击此处（https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-gel-electrophoresis/electrophoresis-chambers/mini-gel-tank/bolt-mini-gel-tank-resources.html）或视频（https://www.youtube.com/watch?v=1FtiX8Skllw），可查看凸轮手柄更换说明。
我们的中型凝胶可使用XCell4 SureLock Midi-Cell进行电泳。
以下是可能原因和解决方案：
膜的转印垫不干净或污染。使用转印垫前，用去污剂浸泡，然后用纯水彻底清洗。转印垫破损或变色后，则更换新的转印垫。
封闭不均匀。孵育皿必须足够大，使封闭液能够完全覆盖膜。每一步都需要摇动或搅动。
以下是可能原因和解决方案：
- 膜被指纹或角蛋白污染：始终佩戴干净的手套并使用镊子来处理膜。处理膜时，仅触碰膜的边缘。
- 二抗浓度较高：按照推荐方法稀释二抗。如果背景仍然很高，但条带强度也很高，则应降低二抗的浓度。
- 一抗浓度较高：降低一抗浓度。
-一抗对蛋白标准品具有亲和力：向蛋白标准品生产商咨询蛋白标准品与一抗的同源性。
- SDS残留或转印后蛋白质与膜的结合较弱：遵循免疫检测前的膜准备说明。
- 封闭时间过短或洗膜时间过长：应确保每一步都达到指定时间。
以下是可能原因和解决方案：
- 封闭不充分或发生非特异性结合：建议尝试使用我们的WesternBreeze封闭剂/稀释液（货号WB7050）。
- 膜污染：仅使用干净的新膜。始终佩戴干净的手套并使用镊子来处理膜。
- PVDF膜本身具有较高的背景：改为使用硝化纤维素膜。
- 硝化纤维素膜未完全湿润：遵循预润湿膜的说明。
- 印迹膜显色过度：遵循推荐的显色时间，当达到可接受的信噪比时，从底物中取出印迹膜。
- 洗膜不充分：遵循推荐的洗膜次数。在一些情况下，可能有必要增加洗膜次数和时间。
- 二抗浓度较高：通过点印迹法确定最佳抗体浓度，必要时可稀释抗体。
- 一抗浓度较高：通过点印迹法确定最佳抗体浓度，必要时可稀释抗体。
以下是可能原因和解决方案：
- 一抗和二抗不匹配：所用二抗应该是针对一抗物种来源的抗体。
- 一抗稀释过度：1) 使用浓度更高的抗体溶液。2) 在4℃孵育更长时间（如，过夜）。3) 使用新鲜的抗体，应注意抗体溶液每使用一次，其有效抗体浓度都会有所降低。
- 封闭液含有可干扰一抗和/或二抗结合的物质：尝试交替使用封闭液± 温和的表面活性剂，如Tween-20（0.01–0.05% v/v）。基于脱脂奶粉、BSA、普通血清、明胶和这些物质的混合物以及其他原料，有多种封闭液配方可用。应注意BSA（1–5%）被认为是硝化纤维素膜的最佳封闭剂。通过点印迹法可轻松检验不同封闭液的效能。
- 一抗不能识别测试物种的相关蛋白质：1) 首先通过点印迹法评估一抗与蛋白质的反应能力。2)检查免疫原序列（如果已提供），确定您的蛋白质中是否含有该序列。3)如果无可用的免疫原序列，则通过PubMed/BLAST比对来评估您的目标蛋白与抗体的目标蛋白之间的同源程度。应注意，许多抗人源蛋白的抗体，也可识别非人源的灵长类动物蛋白，因为人源蛋白与灵长类动物蛋白具有高度的氨基酸同源性。相比之下，许多抗人源蛋白的抗体却不能识别啮齿类动物的相应蛋白（反之亦然）。应记住（注意），序列间显著的同源性并不能保证抗体可识别您的蛋白质。4)尽量每次电泳都使用推荐的阳性对照。
- 与膜结合的蛋白质不足，或样品中的目标蛋白不足：1) 凝胶上每个泳道中的蛋白质上样量至少为20–30 μg（作为起始点），因为，含量低于总蛋白量约0.2%的蛋白质，难以在免疫印迹中被检测到。2) 采用富集步骤以增加目标蛋白的浓度。例如，在转印核蛋白前制备2份细胞核裂解物，或在SDS-PAGE前进行免疫沉淀（IP）。3)减少用于裂解细胞或组织的细胞提取缓冲液的体积。4) 如果需要，应确保蛋白质提取缓冲液中使用新鲜配制的蛋白酶抑制剂和磷酸酶抑制剂。5)尽量使用推荐的阳性对照进行电泳。
- 很少或没有蛋白质转印到膜上：1) 使用可逆性蛋白质染料（如，Invitrogen可逆性膜蛋白质染料、丽春红S、酰胺黑）或预染分子量标准品，检验蛋白质转印效率。2) 确认是否使用了正确的电源极性进行转印。3)应记住，碱性pl值的蛋白质（如，组蛋白）和高分子量蛋白质的转印效果较差。4) 应记住，如果您的目标蛋白分子量较低（≤10 kDa），则转印速度可能比预期更快。5)如果您使用的是PVDF膜，应先将膜在甲醇中预浸泡，然后再浸泡在转膜液中。应注意，转膜液中的甲醇可增加膜与硝化纤维素膜的结合，但减少甲醇可增强高分子量蛋白质的转印效率。6) 低分子量蛋白质可能会穿过孔径为0.45 μm的硝化纤维素膜，因此，应改为使用0.2或0.1μm孔径的NC膜。
- 洗膜或封闭过度：1) 避免过度洗膜。若您的印迹存在其他问题，过度洗膜将导致目标蛋白无法显色。2) 避免由高浓度封闭液成分或较长孵育时间造成的过度封闭。封闭过度可妨碍抗体与蛋白的结合。明胶特别容易遮盖印迹膜上的蛋白质，因此应尽量避免使用。牛奶也会遮盖蛋白质，因此，可尝试使用0.5%牛奶或完全去除牛奶来取代封闭液中的5%牛奶。3)改为使用不同的封闭剂和/或缩短封闭时间。
- 重复使用相同的一抗稀释液 每次免疫印迹都应使用新鲜稀释的抗体，因为每重复使用一次稀释后的抗体，其有效浓度都会有所降低。同时，应记住抗体稀释液的稳定性降低，可能会很快失去活性。
- 与二抗结合的酶失效：1) 每次都使用新鲜稀释的二抗结合物。稀释液中的酶（和抗体）可能会很快失活。2) 若您使用的是辣根过氧化物酶（HRP）标记抗体，则不要在缓冲液中加入叠氮化钠。3)避免高血红素浓度（来自血液污染），否则会干扰基于HRP的检测。4) 避免在含有碱性磷酸酶-抗体结合物的缓冲液中加入磷酸盐，因为磷酸盐会抑制酶活性。
- 您的比色检测或其他检测试剂太旧并且已失活：1) 每次试验均使用新鲜的酶底物。2) 不要使用颜色发生改变或超过有效期的即用型底物试剂。3)除非产品使用手册指示，否则不要稀释底物溶液。
以下是一些建议：
•应确保每个泳道的蛋白上样量均正确——蛋白上样过多可导致条带模糊。
•低比例凝胶不能良好分离条带——尝试使用更高比例的凝胶。
•这可能是由于抗体浓度过高。我们建议遵循生产商的建议进行稀释或确定最佳抗体浓度。
通常，Tricine凝胶的背景染色会稍高于Tris-甘氨酸凝胶。与Tris-甘氨酸凝胶中的对应部分相比，Tricine凝胶中相对较高的溶质浓度减慢了溶液进入凝胶的速度。为了改善这个情况，可通过优化方法延长第二步增敏作用的浸泡时间（可过夜）后再进行后续处理。
若使用Tris-甘氨酸样品缓冲液进行Tricine凝胶电泳，会出现畸形条带且分辨率差。若不小心使用了Tris-甘氨酸电泳缓冲液进行Tricine凝胶电泳，那么，与使用Tris-甘氨酸电泳缓冲液进行Tris-甘氨酸凝胶电泳相比，Tricine凝胶电泳时间更长且分别率较差（特别是较小的蛋白质）。这是由于浓缩胶区域尺寸的增加（甘氨酸是比三甲基甘氨酸更慢的离子），并且Tricine凝胶的离子强度更高。
一种可能的解释为蛋白质样品在电泳完毕之前发生再氧化。在Tricine系统中，还原型样品倾向于发生氧化。加入更多的还原剂并不能解决这个问题。一种方法是使用20 mM DTT在70℃下加热样品30分钟，再使用50 mM碘乙酸，使样品烷基化。另一种抑制氧化的方法是在电泳缓冲液中加入巯基乙酸。该方法的参考文献为Hunkapiller et al., Methods in Enzymology, (91), 399, 1983。使用该方法时应谨慎，因为巯基乙酸既有毒性，又很昂贵。此外，必须使用新鲜的TGA，因为TGA会随时间发生自氧化，并促进样品的再氧化。
Tricine凝胶转印时，我们推荐使用含20%甲醇的1X Tris-甘氨酸转膜缓冲液。Tris-甘氨酸转膜缓冲液可干扰蛋白质测序。因此，如果您想进行蛋白质测序，我们推荐使用无甘氨酸的转膜缓冲液，如1X NuPAGE转膜缓冲液、0.5X TBE转膜缓冲液或CAPS缓冲液（（10mM CAPS（3-环己胺，1-丙磺酸），10%甲醇，pH 11.0）。
不同上样孔规格的凝胶的推荐上样体积和蛋白质上样量见Invitrogen预制胶电泳指南（https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf）第8页。
在SDS发挥的变性作用不充分时，向样品和电泳缓冲液中加尿素，与SDS共同作用，可能会改善样品的溶解性。这必须经过对目标蛋白质的经验验证。
可以。与甘氨酸不同，三甲基甘氨酸不会干扰测序试剂。
不含有，三甲基甘氨酸实际上来自于电泳缓冲液。
Tricine凝胶系统于1987年被Schagger和von Jagow首次描述，是Laemmli Tris-甘氨酸系统的改良版，对较小的蛋白质和多肽具有较好的分离效果。在Laemmli系统中，蛋白质“堆积”在多孔的上半部分凝胶（浓缩胶）中，并存在于由凝胶缓冲液提供的高迁移率的“先导”氯离子与电泳缓冲液中提供的慢低泳动率的“尾随”甘氨酸离子边界之间。这些堆积的蛋白质条带在到达分离胶后开始筛分，根据大小进行分离。但是，浓缩胶中十二烷基硫酸盐（DS）离子（来自SDS样品和电泳缓冲液）的连续积累会阻碍较小蛋白质（低于10 kDa）的分离。DS堆积可导致DS离子与较小蛋白质发生对流混合，导致条带模糊和分辨率降低。DS离子与较小蛋白质的混合也会干扰后续的固定和染色步骤。
为解决该问题，我们提供了以Schagger和von Jagow 改良的Tris-甘氨酸系统为基础而开发的Invitrogen Tricine凝胶系统。这种改进系统采用低pH的凝胶缓冲液，并使用移动更快的三甲基甘氨酸代替甘氨酸作为尾随离子。很多在Tris-甘氨酸系统中随着堆积的DS微粒迁移的小蛋白质和多肽在Tricine凝胶系统中可以很好地与DS离子分离，得到更加清晰的条带和更高的分辨率。
Tricine凝胶中丙烯酰胺：双丙烯酰胺的比值为37.5:1，交联剂的比例为2.6%。
Tricine凝胶包含长度约为8-9 mm的4%浓缩胶。
Tricine凝胶不含SDS。为了获得最佳电泳效果，Tricine系统要求样品和电泳缓冲液中含有SDS，。Tricine凝胶电泳使用Tricine SDS样品缓冲液和Tricine SDS电泳缓冲液。
Invitrogen Tricine凝胶适用于分离多肽和低分子量蛋白（低于10 kDa）。与Tris-甘氨酸凝胶不同，Tricine凝胶可分离分子量低至2 kDa的蛋白质。与甘氨酸(Glycine)不同，三甲基甘氨酸(Tricine)不会干扰测序，所以Tricine凝胶是转印至PVDF膜后直接测序的极佳选择。Tricine凝胶除了具有良好的转印效率，其较低的pH还可将不必要的蛋白质修饰降至最低。Tricine凝胶只能在变性条件下电泳。
Invitrogen Tricine凝胶的推荐保存温度为4℃，在此温度下保质期为4-8周，时间长短取决于凝胶浓度。浓度越高，保质期越短。
以下是出现波浪形染色条带的可能原因和解决方案：
1. 缓冲槽内外的缓冲液高度不同：两个电泳槽中的缓冲液必须全都加至电极的高度。这样不仅可以防止缓冲液从内向外泄漏，还能起到散热的作用，从而防止出现波浪形染色条带。
2. 所用电泳缓冲液稀释度大于1X：我们推荐使用1X电泳缓冲液。
3. 使用旧的电泳缓冲液：应确保使用新鲜的电泳缓冲液，并且不要重复利用电泳缓冲液。
可能是因为Bolt凝胶盒插反了（大塑料板朝前，上样孔朝后），尽管这很难实现。当凝胶反向插入时，电流从凝胶底端流向顶端，导致样品反向电泳。对调导线将转换凝胶电泳方向，但会导致电流从阳极流向阴极。阴极是由带有铂涂层的不锈钢制成，阳极是由铂丝制成。电子从阳极流向阴极，会导致钢芯生锈。但是，当正确连接导线时，电子从阴极流向阳极，铂丝作为电子受体则不会生锈。
注意：当凝胶盒正确插入时，凝胶盒上的印刷字体（凝胶类型、SKU和有效期）是从左往右读的（请参见说明书第11页（https://tools.thermofisher.com/content/sfs/manuals/mini_gel_tank_man.pdf)）。
以下是可能原因和解决方案：
1.使用了浓度过高或错误的缓冲液：检查缓冲液配方；必要时，稀释或重新配制
2. 电流设置过高：将电源条件降低至推荐的电泳条件
以下是可能原因和解决方案：
1. 凝胶盒底部留有胶带：从凝胶盒底部移除胶带。
2. 未完全与电源连接：使用电压表检测所有连接处的电导率。
3. 缓冲液不足：确保电泳槽中的缓冲液足以浸没凝胶上的上样孔。
这可能是由于：
•孔中有碎片
•样品含盐量高（确保盐浓度不超过50–100 mM）
•电泳缓冲液存在问题
•制胶错误
这可能是由凝胶聚合问题和错误的样品制备（最终样品稀释度低于1X）所致。请尝试使用不同批次的相同凝胶，并确保样品正确制备。
可能原因：
还原剂过多（β-巯基乙醇）
皮肤蛋白污染物（角蛋白）
解决方案：
即将上样前，在平衡缓冲液中加入碘乙酰胺，该方法已被证明能消除这种人为条带。
处理凝胶和上样时，使用新鲜的电泳溶液并戴手套。使用高度敏感的染料时，更易出现这种问题。
可能原因：
•上样错误，导致样品残留污染了相邻孔
•电泳缓冲液污染
•凝胶灌制错误：畸形孔
解决方法：
•使用凝胶上样器将样品加到孔中
•减少上样体积
•不要延迟上样
•不要延迟电泳，因为蛋白质会水平扩散；满孔与空孔相邻时，满孔会随时间推移而逐渐污染空孔。
可能原因为：
•上样孔周围的聚合较差
•样品的盐浓度较高
•凝胶界面不均匀
•凝胶安装到夹子上时，对凝胶板造成的压力过大
•凝胶加热不均匀
•凝胶中有不溶物质或整块凝胶上的孔径不一致
•电泳时有气泡
解决方法：
•采用透析、Sephadex G-25或任何其他脱盐柱或使用Amicon浓缩管去除过多的盐或其他物质。
•电泳时，使用冷却装置或降低电流。
部分蛋白质样品可能在电泳过程中再氧化，或在电泳前未完全还原。我们推荐使用新鲜的β巯基乙醇或二硫苏糖醇（DTT）制备新鲜的样品溶液。对于NuPAGE凝胶，我们推荐在电泳缓冲液中加入抗氧化剂。
凝胶脱离凝胶盒的原因可能是：
•过期的凝胶发生降解。
•凝胶保存方式不恰当。
•电泳期间，电流过大导致过多的热量积累。
•聚丙烯酰胺聚合不充分。
鬼带通常被认为是由于凝胶从盒中轻微脱离（lift），导致一些样品流出到其正常迁移点之外。然后它积累起来显示为微弱的第二条带。
出现“微笑”条带可能是因为凝胶中的丙烯酰胺分解，使蛋白质迁移的基质变少。我们建议您确认使用的凝胶未超过有效期。
杠铃形条带可能是由上样量过大所致。当上样量很大时，一部分样品会扩散到孔的边缘。电泳开始后样品通过浓缩胶部分，样品不完全浓缩会使扩散到孔边缘的部分样品出现轻微滞后。较大的蛋白质在低浓度丙烯酰胺的浓缩胶中迁移阻力更大，会加剧这种效应。为缓解这一问题，我们推荐浓缩蛋白质并减少上样量。这会形成“较薄的”起始区域。
以下是可能原因和解决方案：
1. 上样量太大：上样量不要过大
2. 还原剂不新鲜：上样前正确还原样品，不要使用保存在还原剂中的样品
3. 电泳过程中，蛋白质再氧化：使用NuPAGE凝胶电泳时，在电泳缓冲液中加入抗氧化剂
4. 存在高度疏水性区域，在此区域内蛋白质排斥SDS：上样时，使用2X样品缓冲液代替1X
5. 样品含盐过多：沉淀，并使用低盐缓冲液重悬
6. 样品中SDS不足：在阴极槽加SDS（尝试0.1%、0.2%、0.3%和0.4%）
尽管我们推荐使用NuPAGE样品还原剂以加强稳定性，但新鲜的β-巯基乙醇可以代替NuPAGE样品还原剂并获得相同的结果。通常，终浓度为2–5%的β-巯基乙醇足以还原样品。
不能。NuPAGE抗氧化剂在其他凝胶系统的高pH环境下无效。
中型凝胶可使用以下方法转印：
•iBlot干转系统，结合使用Transfer Stacks转印膜组
•Invitrogen半干转仪，最多同时转印2块中型凝胶
•Thermo Scientific Power Blotter，最多同时转印2块中型凝胶
•Thermo Scientific G2Fast Blotter（将于当前库存售尽后停止供应）
所有去污剂，甚至是细胞提取物中的磷脂，都会与SDS形成混合胶团并向下迁移到凝胶中。它们还会干扰SDS与蛋白质的结合平衡。大部分非离子洗涤剂，包括NP-40，是SDS-PAGE最严重的干扰物质。使这种不良影响最小化的经验方法是，将SDS与脂质或其他去污剂的比例保持在10:1或更大。
所有Invitrogen蛋白质凝胶都含有蔗糖。蔗糖是一种密度调节剂，可促进凝胶的灌制。在Invitrogen凝胶电泳上的蛋白质样品会被大量蔗糖污染。因此，不推荐将Invitrogen凝胶用于此应用。
凝胶塑料卡的材料是苯乙烯共聚物。
我们不推荐回收凝胶塑料卡，因为凝胶塑料卡的化学涂层在融化时会产生有毒烟雾，并可能导致污染。
中型凝胶比小型凝胶更宽，因此，每块凝胶的上样孔更多，可容纳更多的样品。在小型凝胶上开展的实验可轻松放大到相同化学成分的中型凝胶上。请在下表中查看不同化学成分Invitrogen小型和中型凝胶的尺寸：
中型凝胶
NuPAGE Bis-Tris、NuPAGE Tris-Acetate和Invitrogen Tris-甘氨酸：凝胶尺寸 13 cm x 8.3 cm，凝胶塑料卡尺寸 15 cm x 10.3 cm
小型凝胶
NuPAGE Bis-Tris、NuPAGE Tris-Acetate和Invitrogen Tris-甘氨酸：凝胶尺寸8 cm x 8 cm，凝胶塑料卡尺寸 10 cm x 10 cm
新型Bolt Bis-Tris Plus（货号NWxxxxBOX）：凝胶尺寸8 cm x 8.3 cm，凝胶塑料卡尺寸 10 cm x 10 cm
老款 Bolt Bis-Tris Plus（货号BGxxxxBOX）： 凝胶尺寸8 cm x 8.3 cm，凝胶塑料卡尺寸 10 cm x 10.5 cm
我们所有的Invitrogen预制蛋白质凝胶（Invitrogen凝胶、NuPAGE凝胶和Bolt Bis-Tris Plus凝胶）都具有小型规格（凝胶塑料卡：10 cm x 10 cm；凝胶：8 cm x 8 cm）。请注意，老款Bolt Bis-Tris Plus小型凝胶（2014年12月31日起停产）的尺寸略有不同（凝胶塑料卡：10 cm x 10.5 cm；凝胶：8 cm x 8.3 cm）。
我们的NuPAGE Bis-Tris、NuPAGE Tris-Acetate和Invitrogen Tris-甘氨酸凝胶也具有较宽的中型规格。注意，Bolt Bis-Tris Plus凝胶无中型规格。
我们的Thermo Scientific Precise预制胶只有小型规格。
小型凝胶
NuPAGE Bis-Tris、NuPAGE Tris-Acetate和Invitrogen Tris-甘氨酸：凝胶尺寸8 cm x 8 cm，凝胶塑料卡尺寸 10 cm x 10 cm
Bolt Bis-Tris Plus（货号NWxxxxBOX）：凝胶尺寸8 cm x 8.3 cm，凝胶塑料卡尺寸 10 cm x 10 cm
老款 Bolt Bis-Tris Plus（货号BGxxxxBOX）： 凝胶尺寸8 cm x 8.3 cm，凝胶塑料卡尺寸 10 cm x 10.5 cm
Thermo Scientific Precise Tris-甘氨酸：凝胶尺寸6.8 cm x 8 cm，凝胶塑料卡尺寸8 cm x 10 cm或凝胶尺寸 8.8 cm x 8 cm， 凝胶塑料卡尺寸10 cm x 10 cm
Thermo Scientific Precise Tris-HEPES ：凝胶尺寸 5.8 cm x 8 cm，凝胶塑料卡尺寸8.5 cm X 10 cm
中型凝胶
NuPAGE Bis-Tris、NuPAGE Tris-Acetate和Invitrogen Tris-甘氨酸：凝胶尺寸 13 cm x 8.3 cm，凝胶塑料卡尺寸 15 cm x 10.3 cm
我们所有的Invitrogen蛋白质凝胶都具有小型规格。某些化学成分的凝胶（NuPAGE Bis-Tris、NuPAGE Tris-Acetate和Invitrogen Tris-甘氨酸凝胶）还具有较宽的中型规格。应注意，Bolt Bis-Tris凝胶无中型规格。我们的Thermo Scientific Precise预制胶只有小型规格。
如果你是在恒定电压下运行凝胶，你不需要根据凝胶的数量增加电压。然而，所观察到的电流和瓦特数将与凝胶数线性相乘。请记住，您的凝胶的预期总电流不应超过电源的电流限制，否则电流将趋于平稳，运行速度将减慢。(例如:使用MES缓冲液运行NuPAGE Bis-Tris凝胶的推荐恒压为200 V，起始电流为110-125 mA/gel，结束电流为70-80 mA/gel。如果电源的电流限制为500毫安，则在满电的情况下可以同时运行的NuPAGE Bis-Tris凝胶的最大数量为500毫安/125毫安 = 4块凝胶。任何额外的凝胶将减少每块凝胶上的电流并增加运行时间。
我们不推荐在同一块凝胶上同时跑还原型和非还原型蛋白质样品，特别是在相邻的泳道中。因为，还原剂可能对距离很近的非还原型样品产生后遗效应。
我们不推荐长期保存还原型蛋白质样品，即使是冷冻保存。因为，样品在保存期间可能发生再氧化，使结果不一致。
请参见以下信息：
Tris-甘氨酸凝胶（除了4% Tris-甘氨酸凝胶）：丙烯酰胺：双丙烯酰胺的比值为 37.5:1 ，交联剂的百分比为2.6%。
4% Tris-甘氨酸凝胶：丙烯酰胺：双丙烯酰胺的比值为76:1， 交联剂的百分比为1.3% 。
在包括Bolt Bis-TrisPlus凝胶在内的大部分凝胶中，浓缩胶为4%。NuPAGE Tris-Acetate凝胶含3.2%浓缩胶。
我们的Invitrogen预制蛋白质凝胶包含长度约为8-9 mm的浓缩胶（正好到达凝胶塑料卡第一嵴线的上方）。使用的生产方法使浓缩胶和分离胶之间形成了一个肉眼无法看到的界面。
•Invitrogen Tris-甘氨酸和InvitrogenTricine小型凝胶：Invitrogen预制胶电泳指南（https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf），第8页
•NuPAGE Tris-Acetate和NuPAGE Bis-Tris小型凝胶：NuPAGE技术指南（https://tools.thermofisher.com/content/sfs/manuals/nupage_tech_man.pdf），第10页
•Bolt Bis-Tris Plus小型凝胶：点击此处（https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-gel-electrophoresis/protein-gels/bolt-bis-tris-gels.html）查看
•Thermo Scientific Precise Tris-HEPES凝胶：Precise Tris-HEPES凝胶使用手册（https://tools.thermofisher.com/content/sfs/manuals/MAN0011499_Precise_Protein_Gels_UG.pdf），第1页
•中型凝胶（Invitrogen Tris-甘氨酸、NuPAGE Bis-Tris和NuPAGE Tris-Acetate）：Invitrogen中型凝胶系统使用手册（https://tools.thermofisher.com/content/sfs/manuals/Invitrogen_midigel_man.pdf），第4页
•Thermo Scientific Precise Tris-甘氨酸凝胶：Precise Tris-甘氨酸凝胶使用手册（https://tools.thermofisher.com/content/sfs/manuals/MAN0011814_Precise_TrisGlycine_Gels_UG.pdf），第1页
Coupling of chromophores to proteins affects the apparent molecular weight of proteins in SDS-PAGE relative to unstained standards. The apparent molecular weight of prestained protein standards is calibrated in the classical TRIS glycine-SDS Laemmli system, however prestained proteins may have different mobility in other electrophoresis buffer and gel systems. It should also be noted that the sizing of proteins by gel electrophoresis does not give an exact value and depends on the protein sequence and post-modification.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The upper bands of the ladder may be degraded by proteases. Ladder, gel, buffer, pipettes, pipette tips, or equipment can be contaminated by proteases during usage. A general recommendation would be to avoid working with proteases in the same room. We would recommend preparing fresh solutions, cleaning the equipment, and using clean pipettes and tips. If the ladder itself is contaminated, please use a new tube of the ladder.
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No, proteins in Thermo Scientific protein ladders are not His tagged. However, non-specific interaction between the ladder proteins and primary or secondary antibodies is possible and some His-Tag detection systems, such as Thermo Scientific 6xHis Protein Tag Stain Reagent Kit, show non-specific interaction. The protein ladder bands are more readily detected when using high antibody concentrations. The non-specific cross-reactivity is difficult to predict, it often has a different pattern dependent on the antibodies used in each individual experiment. The most general way to handle this problem would be to use lower concentrations of antibodies and to use lower amount of protein ladders. It may also be useful to leave one empty well between the ladder and the sample to overcome a possible leakage of the signal to the nearby sample lane.
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PageRuler Unstained protein ladders can be detected directly on Western blots by using Strep-Tactin conjugates or an antibody against the Strep-tag II sequence. All PageRuler and Spectra ladder proteins contain an integral Strep-tag II sequence, however the prestained ladders cannot be detected by Strep-Tactin conjugates.
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All PageRuler and Spectra ladder bands are recombinant prokaryotic proteins purified from E. coli cells. None of them are related to eukaryotic proteins, however this cannot exclude the possibility that the ladder proteins may possess an epitope that is cross-reactive with the antibody used.
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Protein ladder bands can sometimes be detected with chemiluminescent techniques due to non-specific interactions of ladder proteins with either primary or secondary antibodies (or with both). The ladder bands are only rarely detected by chromogenic substrates. The extremely high sensitivity of the chemiluminescent assays is needed to see the bands, so the actual degree of cross-reactivity is low. The non-specific cross-reactivity is difficult to predict, it often has a different pattern depending on the antibodies used. If antibodies recognize a linear epitope, the cross-reactivity may be due to sequence homology. If antibodies react with a denaturation-resistant conformational epitope it could be impossible to identify the exact reason for detected cross-reactivity. The most general way to handle this problem would be to use lower concentrations of antibodies.
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No. Thermo Scientific protein ladders contain a mix of recombinant prokaryotic proteins purified from E. coli cells. E. coli does not have native glycosylation pathways, so none of the ladder proteins are glycosylated.
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Thermo Scientific ladders are not designed for protein quantification. For quantification, we would recommend to use a protein of known concentration as a reference.
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Yes, PageRuler Prestained NIR Protein Ladder (Cat. No. 26635) contains proteins that are blue-stained and fluor-labeled for near-IR fluorescent visualization and protein sizing following electrophoresis.
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Protein ladders can be run on lower percentage gels than we recommend, however it should be expected that several bands of lower molecular weight proteins will run out of the gel if the gel is run until the dye front reaches the bottom of the gel. In case of shorter electrophoresis time it is also possible that some lower bands will not separate and will be covered by the dye front. In addition, lower molecular weight protein bands may look diffused. The lower percentage gels can be used in cases when the customer is interested in visualization of large proteins only.
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Most of the common gel running buffers are composed of Tris-glycine or Tris-tricine. Tris-glycine buffer systems are useful for separation of proteins over a wide range of molecular weights (5-300 kDa) and are compatible with denaturing or non-denaturing conditions. Tris-tricine buffer is generally recommended for the electrophoresis of low molecular weight proteins and peptides (<10 kDa) that need to be reduced and denatured prior to loading. Tris-acetate buffer system is used for separation of larger proteins (>200 kDa).
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Prestained ladders/markers are recommended for approximate determination of molecular weight and for monitoring the progress of the electrophoresis run and the efficiency of protein transfer to membranes during Western blotting procedures. Unstained ladders/markers are used for precise determination of molecular weights in any denaturing buffer system.
We do not provide the exact or approximate concentration of proteins in Thermo Scientific protein ladders, and they are not meant to be used for quantifying the protein concentration of a band. For densitometry assessment, we recommend loading a known amount of a protein standard and determining the linear range according to the gel or membrane stain used.
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DTT is not stable, so it must be added and the reduction performed just prior to loading your samples.
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Precipitation of the LDS or SDS at 4 degrees C is normal. Bring the buffer to room temperature and mix until the LDS/SDS goes into solution. If you do not want to wait for it to dissolve, you can store the sample buffer at room temperature.
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While they are both Bis-Tris based gels, the chemistries are very different since Bolt gels are optimized for western blotting. Another key difference is the wedge well design of the Bolt gels, which allows larger sample volumes to be loaded.
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The neutral operating pH of the NuPAGE Gels and buffers provides following advantages over the Laemmli system:
-Longer shelf life of 8-12 months due to improved gel stability
-Improved protein stability during electrophoresis at neutral pH resulting in sharper band resolution and accurate results (Moos et al, 1998)
-Complete reduction of disulfides under mild heating conditions (70 degrees C for 10 min) and absence of cleavage of asp-pro bonds using the NuPAGE LDS Sample buffer (pH > 7.0 at 70 degrees C)
-Reduced state of the proteins maintained during electrophoresis and blotting of the proteins by the NuPAGE Antioxidant
Please refer to the following paper: Moos M Jr, Nguyen NY, Liu TY (1988) Reproducible High Yield Sequencing of Proteins Electrophoretically Separated and Transferred to an Inert Support. J Biol Chem 263:6005-6008.
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The formulations of buffers for our precast protein gels can be found at this link: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-electrophoresis-buffers-reagents.html
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The Tricine gel system, first described by Schagger and von Jagow in 1987, is a modification of the Laemmli Tris-Glycine system to allow for better resolution of smaller proteins and peptides. In the Laemmli system, the proteins are "stacked" in the porous top portion of the gel (stacking gel) between a highly mobile "leading" chloride ion present in the gel buffer and the slower "trailing" glycine ion supplied by the running buffer. These concentrated, thin bands of protein undergo sieving once they reach the resolving gel, which separates them by size.
The resolution of smaller proteins (under 5 kDa) is hindered by the continuous accumulation of free dodecyl-sulfate (DS) ions (from the SDS sample and running buffers) in the stack. This build-up of DS leads to convective mixing of the DS ions with the smaller proteins, causing fuzzy bands and decreased resolution. The mixing of the DS ions with the small proteins will also interfere with the fixing and staining process later. To solve this problem, Schagger and von Jagow replaced the trailing glycine ion with a faster moving Tricine trailing ion. Many small proteins which run with the stacked DS in the Tris Glycine system will separate from DS in the Tricine gel system, resulting in sharper, cleaner bands and better resolution.
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Either BME or DTT can be used in the NuPAGE LDS Sample Buffer.
Make sure that a fresh solution of BME is used. FINAL concentration:
DTT 50-100 mM
BME 2-5%
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There may be too much beta-mercaptoethanol (BME), sample buffer salts, or dithiothreitol (DTT) in your samples. If the proteins are over-reduced, they can be negatively charged and actually repel each other across the lanes causing the bands to get narrower as they progress down the gel.
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Barbell-shaped bands are a result of loading too large a sample volume.
When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will stack incompletely, causing a slight retardation of the portion of the sample that diffused to the sides of the wells.
This effect may be intensified in larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel.
To alleviate the problem, concentrate the protein and load a smaller volume. This gives a "thinner" starting zone.
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Some potential causes are:
1) Re-oxidation of protein during run
2) Protein has highly hydrophobic regions where protein can exclude SDS.
Steps you can take to improve results:
1) Reduce samples right before loading, and add antioxidant to running buffer. Do not use samples that have been stored in reducing agent.
2) Load sample with 2X sample buffer instead of 1X.
3) Add SDS to upper chamber buffer: try 0.1, 0.2, 0.3, and 0.4% (don't go any higher than 0.4%)
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Yes. All detergents and even phospholipids in cell extracts will form mixed micelles with SDS and migrate down into the gel.
They can also interfere with the SDS:protein binding equilibrium. Most of the nonionic detergents significantly interfere with SDS-PAGE.
We recommend that you keep the ratio of SDS to lipid or other detergent at 10:1 (or greater) to minimize these effects.
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There shouldn't be any negative effects unless the percentage of acetonitrile reaches 40% or 50% of the sample volume.
At these concentrations, there is the possibility of the acetonitrile affecting the binding of SDS to the protein, which, in turns, affects the migration of the protein.
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There is no SDS in the gels. Denaturing conditions are created by using sample buffers and running buffers that contain SDS.
The benefit of not having SDS in the gels is that the gel can be used for both native and denaturing conditions.
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Invitrogen gels are packaged in Packaging Buffer: Tris HCl, pH 8.65, with 0.02% sodium azide (expect that residual acrylamide monomer is also present). Wear gloves at all times when handling gels.
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A temperature increase to 35°C to 40°C during electrophoresis is not uncommon for Tricine gels. If you want to run the gels at a cooler temperature, the lower (outer) buffer chamber can be filled higher or they can be run at a lower voltage, for example 100 V.
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For non-sequencing applications, any transfer buffer used with Tris-Glycine gels can be used with Tricine gels including Tris-Glycine transfer buffer. For sequencing applications, the buffer should be chemically compatible with sequencing protocols. Non-glycine based transfer buffers such as the NuPAGE Transfer buffer, 1/2X TBE Transfer buffer, or CAPS Buffer can be used for N-terminal sequencing . Generally, a pH which is close to neutral is desirable to maintain gel and protein stability. High current should be avoided because it can lead to heat generation and instability.
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If the Tricine gel is run with Tris-Glycine sample buffer, the bands will behave abnormally and resolve poorly. If the Tricine gel is accidentally run with Tris-Glycine running buffer, the gel will take longer to run and the resolution, especially for smaller proteins, will be worse than when the proteins are run on a Tris-Glycine gel with Tris-Glycine buffers. This is due to a combination of increase in stack area size (glycine is a slower ion than Tricine) and the higher ionic strength of the Tricine gel.
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Protein samples are possibly reoxidizing before the run is complete in the Tricine gel system. Since Tricine is a glycine derivative, the running pH ranges of the two systems are different. As a consequence, reduced samples tend to oxidize more in the Tricine system. Adding more reducing agent will not solve the problem.
One option is to alkylate the sample by reducing with 20 mM DTT at 70°C for 30 min, followed by 50 mM iodoacetic acid to alkylate.
Another method which inhibits oxidation is the addition of thioglycolic acid (TGA) to the running buffer. The reference to this is described by Hunkapiller et al, Methods of Enzymology, (91), 399, 1983.
Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to become oxidized itself over time. Oxidized TGA will actually promote sample re-oxidation.
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TCEP, Tris Carboxy Ethyl Phosphene is an alternative sulfhydryl reducing agent for protein samples. It is an extremely potent and effective reducing agent for particularly ‘difficult' proteins. It is compatible with the Tris-Glycine gels and NuPAGE gels. It should be added to the sample buffer for these systems. 20 mM final (maximum) concentration is sufficient for samples. You may add alkylating agents, e.g. Iodine (50 mM Iodoacetic acid), to prevent re-forming of S-S bonds but it is not necessary. Do not heat because this will hydrolyze much of your sample. Instead let the sample sit for several minutes at RT and then load.
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If the Antioxidant is omitted from the running buffer, it is possible to resolve reduced and non-reduced samples on the same gel, although the resolution may be lower. Furthermore, it is not recommended that the reduced and non-reduced samples be run side-by-side in adjacent lanes.
However, because of the neutral pH of the NuPAGE gels, the reducing agent (beta-mercaptoethanol or DTT) will not migrate through the gel with the protein the way it does in the basic environment of the Tris-Glycine gels. Instead, the reducing agent tends to remain at the top of the gel. For this reason, the NuPAGE Antioxidant is incorporated into the buffer in the upper buffer chamber. The antioxidant is able to migrate fully with the proteins and keep them reduced. As a result, it is possible that proteins prepared as non-reduced samples could become somewhat reduced during the electrophoresis run. This would result in smearing of the samples.
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Here are possible causes and solutions:
- Not enough volume of ladder loaded on the gel: Load an appropriate volume of the ladder onto the gel. Here are our recommendations:
--- Mini-gel: 5 µL per well (0.75-1.0 mm thick) or 10 µL per well (1.5 mm thick)
--- Large gel: 10 µL per well (0.75-1.0 mm thick) or 20 µL per well (1.5 mm thick)
- Incomplete or poor transfer: Optimize transfer conditions
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Here are possible causes and solutions:
Here are possible causes and solutions:
- Ladder was boiled: Discard boiled aliquot.
- Too much volume of ladder used: Add less volume or dilute the ladder in protein loading buffer prior to use.
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The fading is most likely due to detergent in the western blocking/washing solutions that can remove some of the proteins from the membrane. The dye itself will not wash off of the proteins because it is covalently bound. We have found that smaller pore size membranes retain the proteins better during blocking and wash procedures, and hence recommend use of 0.2 µm instead of 0.45 µm membranes for best resolution and protein retention. After transfer, it is a good idea to circle the pre-stained bands with a pencil on the membrane, so band positions can be identified after blocking and processing.
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- Decrease voltage, current or length of transfer time
- Make sure that the methanol concentration in the transfer buffer is proper; use a methanol concentration of 10-20% methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane.
- Make sure that the SDS concentration (if added) in the transfer buffer is proper, don't use more than 0.02-0.04% SDS. Using too much SDS can prevent binding of proteins to the membrane.
- Check the pore size of the membrane and the size of the target protein. Proteins smaller than 10 kDa will easily pass through a 0.45 µm pore size membrane. If proteins smaller than 10 kDa are of interest, it would be better to use a 0.2 µm pore size membrane.
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- Increase voltage, current or length of time for transfer
- SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes. This inhibition is higher for nitrocellulose than for PVDF. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. We recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing 10% methanol and 0.01%SDS.
- Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Make sure that the methanol concentration in the transfer buffer is not more than 10-20% and that high-quality, analytical grade methanol is used.
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Pre-stained standards have a dye that is covalently bound to each protein that will result in the standard migrating differently in different buffer systems (i.e., different gels). As a result, using a pre-stained standard for molecular weight estimation will only give the apparent molecular weight of the protein. Pre-stained standards may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained standard should be used.
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- While loading, take care to make sure that there is no cross-contamination from adjacent sample lanes.
- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can result in extra bands and this is a problem especially with silver-stained gels.
- Improper storage of the standard or repeated freeze/thawing can result in protein degradation.
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Here are some suggestions:
- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can cause smearing and this is a problem especially with silver stained gels.
- Bands will not be as well resolved in low percentage gels. Try using a higher percentage gel.
- If the bands look smeary and non-distinct after a western transfer/detection, this may be due to the antibody being too concentrated. Follow the manufacturer's recommended dilution or determine the optimal antibody concentration by dot-blotting.
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Here are some suggestions:
- Check the gel type/percentage of the gel that was used. Depending on the gel type and/or percentage, all the bands may not be seen. For example, the smallest bands of the protein standard may not resolve on a very low percentage gel whereas the higher molecular weight bands may not resolve on a high percentage gel.
- Check the expiration date on the protein standard. Expired lots may result in faded or missing bands due to protein degradation.
- Check the storage conditions for the protein standard. Improper storage conditions will compromise the stability of the proteins in the standard.
- Make sure that the protein standard was not heated/boiled prior to loading on the gel. Our protein standards are ready to load and we do not recommend heating/boiling them as this may cause degradation of proteins in the standard.
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The Spectra Protein Ladders contain recombinant prokaryotic proteins and do not contain any animal-derived proteins.
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The Spectra Multicolor Low Range Protein Ladder is a prestained mixture of six recombinant proteins (1.7 to 40 kDa) for use in gel electrophoresis and western blotting. Three different chromophores are bound to the proteins, producing a brightly colored ladder.
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We recommend storing the Spectra Protein Ladders at -20 degrees C where they are stable for a year. The Spectra Multicolor Broad Range Protein Ladder and Spectra Multicolor High Range Protein Ladder are stable for up to 3 months at 4 degrees C. The Spectra Multicolor Low Range Protein Ladder is stable for up to 2 months at 4 degrees C.
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Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is prestained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.
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Our protein standards are not designed for protein quantitation.
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We do not recommend using our prestained standards for native gel electrophoresis since they are already denatured (in SDS sample buffer) and pre-reduced (by a proprietary method), and will not resolve well in under native conditions.
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We recommend using unstained protein ladders for molecular weight estimation applications as prestained ladders have a dye that is covalently bound to each protein that will result in the ladder migrating differently in different buffer systems (i.e., different gels). As a result, using a prestained ladder for molecular weight estimation will only give the apparent molecular weight of the protein. Prestained ladders may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained ladder should be used.
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Please find this information in the respective manuals for the individual protein standards.
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Our protein standards are ready to load. We do not recommend heating them as this may cause protein degradation.
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Except for our NativeMark Unstained Protein Standard (designed for native electrophoresis), all of the other unstained and prestained standards we offer (Invitrogen Sharp, SeeBlue, SeeBlue Plus2, BenchMark, HiMark) have been pre-reduced (by a proprietary method). Hence, you do not need to add reducing agent.
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The Mini Blot Module is designed exclusively for the Mini Gel Tank. It will also fit in the Bolt Mini Gel Tank (discontinued as of December 31, 2014) but will not fit in the XCell SureLock Mini Cell or other vendors' electrophoresis tanks.
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Due to the universal electrode design, the Mini Blot Module fits on either side of the Mini Gel Tank.
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Yes, we offer the Mini Blot Module (Cat. No. B1000), designed to be used with the Mini Gel Tank. This blot module will also work with the Bolt Mini Gel Tank (discontinued as of December 31, 2014). Please note that the Bolt Mini Blot Module (discontinued as of December 31, 2014) is also compatible with both the Bolt Mini Gel Tank and the Mini Gel Tank.
Bolt Bis-Tris Plus gels can also be transferred using the XCell SureLock Mini Cell, iBlot Dry transfer system, or using the Invitrogen Semi-Dry Blotter.
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Chlorobutanol is used as a preservative in the NuPAGE transfer buffer and is not necessary for efficient transfer of proteins. You may prepare the buffer without chlorobutanol but keep in mind that the buffer will not be stable for long periods. We recommend using it within 2 weeks.
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We do not recommend using Carbonate or CAPS transfer buffers to transfer NuPAGE gels as the transfer efficiency will be badly compromised. Further, the high pH environment (>pH 9) of these buffers will make the NuPAGE Antioxidant non-functional.
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To increase efficiency of transfer of high molecular weight proteins from NuPAGE gels, we recommend pre-equilibrating the gel in 2x NuPAGE Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x NuPAGE transfer buffer containing methanol and 0.01% SDS.
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We recommend marking the cassette at the bottom of the wells with a marker pen prior to placing the cassette in the electrophoresis tank.
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Here are possible causes and solutions:
- Tape left on the bottom of the cassette. Remove tape from bottom of cassette.
- Connection to power supply not complete. Check all connections with a voltmeter for conductance.
- Insufficient buffer level. Make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.
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Here are possible causes and solutions:
- Buffers are too concentrated or incorrect. Check buffer recipe; dilute or re-make if necessary.
- Current is set at a higher limit. Decrease current to recommended running conditions (see Page 8 of the manual).
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Here are possible causes and solutions:
- Buffers are too dilute. Check buffer recipe; remake if necessary.
- Buffer chamber is leaking. Make sure the cassette clamp is firmly seated, the gaskets are in place and the cassette clamp is locked.
- Current is set too low. Set correct current.
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Here are the replacement parts we offer for the Mini Gel Tank:
Replacement part - Cat. No.
Gel Runner Tank - B4478641
Mini Gel Tank Lid - A25944
Mini Gel Tank Base - A25950
Cassette Clamp, left - A25946
Cassette Clamp, right - A25945
Cassette Clamp Cam Handle Set - A26732
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Here are our recommendations:
- When electrophoresis is complete, dispose of the buffer appropriately. Rinse the tank with water to remove residual buffer.
- Clean the surface of the Mini Gel Tank with a soft non-abrasive, lint-free cloth dampened with water.
- Do not use harsh detergents or solvents to clean the unit.
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The Mini Gel Tank is not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene) or acetone.
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The Precise Tris-HEPES gels are compatible with the XCell SureLock Mini Cell or Mini Gel Tank when used with adaptor plates.
Note: Two adaptor plates are required when running just one gel and one adaptor plate is required when running two gels using the XCell SureLock Mini Cell or Mini Gel Tank.
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Our Original Bolt Bis-Tris Plus Mini gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) can only be run in the Bolt Mini Gel Tank (discontinued as of December 31, 2014, and will be offered until inventory is depleted).
Our New Bolt Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX), as well as our Invitrogen Mini gels and NuPAGE Mini gels can be run using the Mini Gel Tank, or XCell SureLock Mini-Cell. To run these gels using the Bolt Mini Gel Tank (discontinued as of December 31, 2014), upgrading of the tank is necessary by replacing the black 10.5 cm cassette clamp cam handles with gray 10 cm cassette clamp cam handles (Cat. No. A26732, Cassette Clamp Cam Handle Set). Instructions for replacement of the cam handles can be found here (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gel-electrophoresis-chamber-systems/mini-gel-tank/resources-upgrading-bolt-mini-gel-tank.html).
Our Midi gels can be run using the XCell4 SureLock Midi-Cell.
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Here are possible causes and solutions:
- Membrane blotting pads are dirty or contaminated. Soak pads with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Blocking was uneven. The incubation dish must be sufficiently big to allow thorough coverage of membrane. Shake or agitate during each step.
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Here are possible causes and solutions:
- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.
- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.
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Here are possible causes and solutions:
- Insufficient blocking or non-specific binding: We suggest trying our WesternBreeze Blocker/Diluent (Cat. No. WB7050).
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Blot is overdeveloped: Follow recommended developing time and remove blot from substrate when signal - to -noise ratio is acceptable.
- Insufficient washing ; Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentrated secondary antibody used: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
- Concentrated primary antibody used: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
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Here are possible causes and solutions:
- The primary antibody and secondary antibody are not compatible: Use a secondary antibody that was raised against the species in which the primary antibody was raised.
- The primary antibody is too dilute: 1) Use a more concentrated antibody solution. 2) Incubate longer (e.g., overnight) at 4 degrees C. 3) Use fresh antibody and keep in mind that each time an antibody solution is used, its effective antibody concentration decreases.
- Something in your blocking buffer interferes with binding of the primary and/or secondary antibody: Try an alternate blocking buffer ± a mild surfactant like Tween-20 (0.01-0.05% v/v). There are many blocking buffer recipes available, based on non-fat dry milk, BSA, normal serum, gelatin and mixtures of these and other materials. Note that BSA (1-5%) is considered the best blocker for nitrocellulose membranes. It is easy to check the efficacy of different blocking buffers by performing dot-blots.
- The primary antibody does not recognize the protein in the species being tested: 1) Evaluate primary antibodies by dot-blotting first to how well they react with your protein. 2) Check the immunogen sequence, if provided, and determine if it is found in your protein. 3) If no immunogen sequence is available, perform a PubMed/BLAST alignment to assess the degree of homology between your target protein and the protein against which the antibody was generated. Note that many antibodies against human proteins will also recognize the non-human primate version because there is usually a high degree of amino acid identity. In contrast, many antibodies against human proteins will not recognize the corresponding proteins from rodents (and vice versa). Remember that significant homology between sequences does not guarantee that the antibody will recognize your protein. 4) Always run the recommended positive control, if available.
- Insufficient protein is bound to the membrane or the protein of interest is not abundant enough in the sample: 1) Load at least 20-30 ?g protein per lane on your gels (as a starting point), since proteins representing less than ~0.2% of the total protein are difficult to detect on western blots. 2) Use an enrichment step to increase the concentration of the target protein. For example, prepare two nuclear lysates prior to blotting nuclear proteins or perform an immunoprecipitation (IP) prior to SDS-PAGE. 3) Reduce the volume of cell extraction buffer used to lyse your cells or tissue. 4) Be sure to use freshly prepared protease inhibitors and phosphatase inhibitors, if needed, in your protein extraction buffer. 5) Run the recommended positive control, if available.
- Poor or no transfer of the proteins to the membrane 1) Check the protein transfer efficiency with a reversible protein stain like Invitrogen Reversible Membrane Protein Stain, ponceau S, amido black or use pre-stained molecular weight standards. 2) Verify that the transfer was performed with the correct electrical polarity. 3) Remember that proteins with basic pI values (e.g., histones) and high MW may not transfer well. 4) Remember that if your target protein has a low MW (≤10 kDa), it may transfer more quickly than expected. 5) If you are using PVDF membranes, make sure to pre-soak the membrane in methanol first before soaking it in transfer buffer. Note that methanol in transfer buffer increases protein binding to nitrocellulose, but omitting methanol can increase transfer efficiency of high MW proteins. 6) Low MW proteins may pass through the 0.45 µm pores in nitrocellulose membranes, so switch to NC with 0.2 or 0.1 µm pores instead.
- Excessive washing or blocking of the membrane:- 1) Avoid over-washing the membrane. Extra washing will not allow you to visualize your protein of interest if there are other problems with your blot. 2) Avoid over-blocking by using high concentrations of the blocking buffer components or long incubation times. Too much blocking can prevent your antibodies from binding to your protein. Gelatin, in particular, can mask proteins on the blot, so avoid it, if possible. Milk can also mask proteins, so instead of using 5% milk in your blocking buffer, try using it at 0.5% instead, or remove it altogether. 3) Switch to a different blocking reagent and/or block the blot for less time.
- Using the same solution of diluted primary antibody repeatedly: Use freshly-diluted antibody for each western blot because the effective concentration of a diluted antibody decreases each time it is re-used. Also, remember that dilute solutions of antibodies are less stable and may lose their activity rapidly.
- The enzyme conjugated to your secondary antibody is not working: 1) Make a fresh dilution of your secondary antibody conjugate each time you need it. Enzymes (and antibodies) may lose activity quickly in dilute solutions. 2) Omit sodium azide in buffers if you are using HRP-conjugated antibodies. 3) Avoid high heme concentrations (from blood contamination), which can interfere with HRP-based detection. 4) Avoid using phosphate in buffers with alkaline phosphatase-antibody conjugates because phosphate inhibits enzyme activity.
- Your colorimetric or other detection reagent is old and inactive: 1) Use fresh enzyme substrate for each experiment. 2) Don't use ready-to-use substrate reagents if they have changed color on their own or if they have passed their expiration date. 3) Do not dilute substrate solutions unless instructed to do so in the product manual.
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Here are some suggestions:
- Make sure that the correct amount of protein is loaded per lane; loading too much protein can cause smearing.
- Bands will not be as well resolved in low percentage gels; try using a higher percentage gel.
- This may be due to the antibody being too concentrated. We recommend following the manufacturer's recommended dilution or determining the optimal antibody concentration
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In general, background staining in Tricine gels is slightly higher than in Tris-Glycine gels. The relatively higher concentration of solutes in Tricine gels as compared to their Tris-Glycine counter parts appears to slow down the rate of solution exchange into the gel. This can be counteracted by increasing the soak time in the second sensitization step (you may leave it in overnight) as per the modified procedure, and then proceed.
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If the Tricine gel is run with Tris-Glycine sample buffer, the bands will behave abnormally and resolve poorly. If the Tricine gel is accidentally run with Tris-Glycine running buffer, the gel will take longer to run and the resolution, especially for smaller proteins, will be worse than when the proteins are run on a Tris- Glycine gel with Tris-Glycine buffers. This is due to a combination of increase in stack area size (glycine is a slower ion than tricine) and the higher ionic strength of the Tricine gel.
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One potential explanation is that the protein sample is getting re-oxidized before the run is complete. Reduced samples tend to oxidize more in the Tricine system. Adding more reducing agent will not solve the problem. One option is to alkylate the sample by reducing with 20 mM DTT at 70 degrees C for 30 minutes, followed by 50 mM iodoacetic acid. Another method which inhibits oxidation is the addition of thioglycolic acid to the running buffer. The reference to this is described by Hunkapiller et al., Methods in Enzymology, (91), 399, 1983. Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to get self-oxidized over time and will promote sample re oxidation.
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For blotting Tricine gels, we recommend using 1X Tris-Glycine Transfer Buffer with 20% methanol. The Tris-Glycine Transfer Buffer interferes with protein sequencing. Hence, if you are performing protein sequencing, we recommend using a non-glycine based transfer buffer such as 1X NuPAGE Transfer Buffer, 0.5X TBE Transfer Buffer or CAPS buffer (10 mM CAPS (3 cyclohexylamino, 1-propanesulfonic acid), 10% methanol, pH 11.0).
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The recommended sample loading volumes and protein loading amounts for the different well formats can be found at:
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/recommended-well-loading-volumes-sample-loads.html.
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Adding urea to the sample and running buffers, in conjunction with SDS, may provide improved solubilization of the sample if denaturation by SDS does not prove to be sufficient. This must be tested empirically for the protein of interest.
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Yes. Tricine, unlike glycine, does not interfere with sequencing reagents.
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No, the Tricine is actually supplied by the running buffer.
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The Tricine gel system, first described by Schagger and von Jagow in 1987, is a modification of the Laemmli Tris-Glycine system to allow for better resolution of smaller proteins and peptides. In the Laemmli system, the proteins are "stacked" in the porous, top portion of the gel (stacking gel) between the highly mobile "leading" chloride ions, present in the gel buffer and the slower "trailing" glycine ions, supplied by the running buffer. These stacked protein bands undergo sieving once they reach the separating gel, thus resolving by size. However, the resolution of smaller proteins (under 10 kDa) is hindered by the continuous accumulation of free dodecyl-sulfate (DS) ions (from the SDS sample and running buffers) in the stacking gel. This build-up of DS leads to convective mixing of the DS ions with the smaller proteins, causing fuzzy bands and decreased resolution. The mixing of the DS ions with the small proteins also interferes with the fixing and staining process later.
To solve this problem, we offer the Invitrogen Tricine gel system that is based on the Tris-Glycine system developed by Schagger and von Jagow. This modified system uses a low pH in the gel buffer and substitutes the trailing glycine ions with faster moving tricine trailing ions. Many small proteins and peptides that migrate with the stacked DS micelles in the Tris-Glycine system are now well separated from DS ions in the Tricine gel system, resulting in sharper, cleaner bands and higher resolution.
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The ratio of acrylamide:bisacrylamide in our Tricine gels is 37.5:1 and percentage of crosslinker is 2.6%.
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Tricine gels contain a 4% stacking gel that is ~8 to 9 mm long.
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Tricine gels do not contain SDS. The Tricine system requires SDS in the sample and running buffers for best results. They are run using the Tricine SDS Sample buffer and Tricine SDS Running buffer.
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Invitrogen Tricine Gels are ideal for peptides and low molecular weight proteins (less than 10 kDa). Unlike Tris-Glycine gels, Tricine gels allow resolution of proteins with molecular weights as low as 2 kDa. Tricine, unlike glycine, will not interfere with sequencing, so Tricine gels are an excellent choice for direct sequencing after transferring to PVDF. In addition to good transfer efficiency, the Tricine system has a lower pH which minimizes unwanted protein modification. Tricine gels can only be run under denaturing conditions.
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The recommended storage temperature for Invitrogen Tricine gels is 4 degrees C where the shelf life varies from 4-8 weeks depending upon the gel percentage. The higher the percentage, the shorter is the shelf life.
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Here are some causes and solutions for wavy dye fronts:
1) Difference in buffer level between the inner and outer buffer chambers: Both buffer chambers must be filled up to the electrode with wells completely covered. This will not only prevent leaks from the inside to the outside but will also act a heat sink and prevent wavy dye fronts.
2) Using running buffer that was diluted more than 1X: We recommend using 1X running buffer.
3) Using old running buffer: Make sure that the running buffer is fresh and don't reuse the running buffer.
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It is likely that the Bolt gel cassette was inserted backwards into the unit (large plate facing the front and the wells facing the back) even though this is pretty difficult to do. When the gel is inserted backwards, the current flows from the bottom of the gel to the top, resulting in the samples running in the opposite direction. Reversing of the leads will switch the direction of the gel run, however, this will cause the current to flow from the anode to the cathode. The cathode electrode is made of stainless steel with platinum coating, and the anode electrode is made of platinum wire. Flow of electrons from the anode to the cathode will result in rusting of the steel core. On the other hand, when the leads are connected properly, the electrons flow from the cathode to the anode and the recipient of the electrons is the platinum wire that does not rust.
Note: When the Bolt gel cassette is inserted properly into the Bolt Mini Gel Tank or Mini Gel Tank, the lettering (gel type, SKU and expiration date) printed on the gel cassette reads from left to right (please see Page 11 of the manual (https://tools.thermofisher.com/content/sfs/manuals/mini_gel_tank_man.pdf).
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Here are possible causes and solutions:
1) Buffers are too concentrated or incorrect: Check buffer recipe; dilute or remake if necessary
2) Current is set at a higher limit: Decrease current to recommended running conditions.
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Here are possible causes and solutions:
1) Tape left on the bottom of the cassette: Remove tape from bottom of cassette.
2) Connection to power supply not complete: Check all connections with a voltmeter for conductance.
3) Insufficient buffer level: Make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.
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This could be due to:
*Debris in the well
*High salt in the sample (make sure that the salt concentration does not exceed 50-100 mM)
*Running buffer issue
*Gel casting error
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This could be due to a gel polymerization issue combined with incorrect sample preparation (final sample dilution less than 1X). Please try a different lot of the same gel and make sure that the sample is correctly prepared.
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Possible cause:
*Excess reducing agent (beta-mercaptoethanol)
*Skin protein contaminants (keratin)
Remedy:
*The addition of iodoacetamide to the equilibration buffer just before applying the sample to the gel has been shown to eliminate these artifact bands.
*Use new electrophoretic solutions and wear gloves when handling and loading the gel. This issue is more common when highly sensitive stains are used.
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Possible cause:
*Carry-over contamination of sample from one well into neighboring wells due to loading error
*Contaminated running buffer
*Gel casting error: malformed wells
Remedy:
*Use a gel loading tip to load wells
*Reduce the sample volume
*Do not delay while loading wells
*Do not delay after the run, as proteins can diffuse horizontally; a full well left next to an empty well would eventually contaminate the empty well over time.
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Possible cause:
*Poor polymerization around sample wells
*High salt concentration in sample
*Uneven gel interface
*Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly
*Uneven heating of the gel
*Insoluble material in the gel or inconsistent pore size throughout the gel
*Air bubble during the run
Remedy:
*Remove excess salt/other material by dialysis, Sephadex G-25 or any other desalting column or using an Amicon concentrator.
*Either use a cooled apparatus or reduce the current at which electrophoresis is performed.
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A portion of the protein sample may have re-oxidized during the run, or may not have been fully reduced prior to the run. We recommend preparing fresh sample solution using fresh beta-mercaptoethanol or dithiothreitol (DTT). For NuPAGE gels, we recommend adding antioxidant to the running buffer.
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Gel lifting off the cassette can be caused by:
*Expired gels that are degrading
*Improper storage of gels
*Too much heat accumulating during the electrophoresis run due to excessive current
*Insufficient polymerization of the polyacrylamide
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Ghost bands are usually attributed to a slight lifting of the gel from the cassette, which results in the trickling down of some sample beyond its normal migration point. It then accumulates and appears as a faint second band.
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"Smiling" bands may be the result of the acrylamide in the gel breaking down, leaving less of a matrix for the proteins to migrate. We recommend checking to ensure that the gels have not been used past their expiration date.
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Barbell shaped bands are a result of loading too large of a sample volume. When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will incompletely stack causing a slight retardation of the portion of the sample that diffused to the sides of the wells. This effect may be intensified for larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel. To alleviate the problem, we recommend concentrating the protein and loading a smaller volume. This gives a "thinner" starting zone.
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Here are possible causes and solutions:
1) Sample overload: Do not overload samples
2) Addition of reducing agent that is not fresh: Reduce samples right before loading and do not use samples that have been stored in reducing agent
3)Re-oxidation of the protein during the run: Add antioxidant to the running buffer if you are running NuPAGE gels
4) Presence of highly hydrophobic regions where the protein can exclude SDS: Load the sample with 2X sample buffer instead of 1X sample buffer
5) Excess salt in the sample: Precipitate and reconstitute in lower salt buffer
6) Not enough SDS in the sample: Add SDS to the upper buffer chamber (try 0.1%, 0.2%, 0.3% and 0.4% SDS)
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Although we recommend using the NuPAGE Sample Reducing agent for stability reasons, fresh, neat beta-mercaptoethanol can be substituted for the NuPAGE Sample Reducing Agent, with equivalent results. A final concentration of 2-5% beta-mercaptoethanol is usually sufficient to reduce the sample.
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No. It is not efficient at the higher pH values of the other gel systems.
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Midi gels can be transferred using:
*iBlot Dry Blotting System in conjunction with Transfer Stacks
*Invitrogen Semi-Dry Blotter for simultaneous transfer of up to 2 Midi-gels
*Thermo Scientific Power Blotter for simultaneous transfer of up to 2 Midi gels
*Thermo Scientific G2 Fast Blotter (will be discontinued as soon as we exhaust current inventory).
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All detergents, or even phospholipids in cell extracts, will form mixed micelles with SDS and migrate down into the gel. They can also interfere with the SDS:protein binding equilibrium. Most of the non-ionic detergents, including NP-40, are the worst at interfering with SDS-PAGE. The rule of thumb is to keep the ratio of SDS to lipid or other detergent at 10:1 or greater to minimize these effects.
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All Invitrogen protein gels contain sucrose as a density-adjusting agent to facilitate pouring of the gel. Protein samples run on Invitrogen gels would be contaminated with large amounts of sucrose. Thus, Invitrogen gels are not recommended for this application.
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The cassettes are made of a styrene copolymer.
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We do not recommend recycling our plastic cassettes because they have a chemical coating on them that may produce toxic fumes when melted and potentially cause contamination.
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Midi gels are wider than Mini gels and hence have a larger number of wells to accommodate additional samples in one gel. An experiment from a Mini gel can be easily scaled-up to a Midi gel of the same gel chemistry.
Midi gels:
*NuPAGE Bis-Tris, NuPAGE Tris-Acetate, & Invitrogen Tris-Glycine: Gel dimensions are 13cm x 8.3cm and Cassette dimensions are 15cm x 10.3cm.
Mini gels:
*NuPAGE Bis-Tris, NuPAGE Tris-Acetate, & Invitrogen Tris-Glycine: Gel dimensions are 8cm x 8cm and Cassette dimensions are 10cm x 10cm.
*New Bolt Bis-Tris Plus (Cat. No. NWxxxxxBOX): Gel dimensions are 8cm x 8.3cm and Cassette Dimensions are 10cm x10cm.
*Original Bolt Bis-Tris Plus (Cat. No. BGxxxxxBOX): Gel dimensions are 8cm x 8.3cm and Cassette Dimensions are 10cm x 10.5cm.
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All of our Invitrogen precast protein gels (NuPAGE gels, Bolt Bis-Tris Plus gels, and Novex gels) are available in Mini format. Our Mini gel dimensions are 8 cm x 8 cm and the cassette dimensions are 10 cm x 10 cm.
Our NuPAGE Bis-Tris, NuPAGE Tris-Acetate, and Novex Tris-Glycine Plus gels are also available in the wider Midi format. Our Midi gel dimensions are 8 cm x 13 cm and the cassette dimensions are 10 cm x 15 cm.
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All our Invitrogen protein gels are available in Mini format. Certain gel chemistries (NuPAGE Bis-Tris, NuPAGE Tris-Acetate, and Invitrogen Tris-Glycine gels) are also available in the wide Midi format.
Note that Bolt Bis-Tris gels are not available in the Midi format and our Thermo Scientific Precise precast gels are only available in Mini format.
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If you are running the gels at constant voltage, you do not need to increase the voltage regardless of the number of gels. However, the resulting current and wattage observed will multiply linearly with the number of gels. Keep in mind that the expected total current for your gels should not exceed the current limit of the power supply, or else the current will plateau and the run will slow down. (For example: Recommended constant voltage for running a NuPAGE Bis-Tris gel with MES Buffer is 200 V, with a starting current of 110-125 mA/gel and end current of 70-80 mA/gel. If the power supply has a current limit of 500 mA, the maximum number of NuPAGE Bis-Tris gels that can be run at one time with full power is 500 mA/125 mA = 4 gels. Any additional gels will decrease the current per gel and increase the run time.
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We do not recommend running reduced and non-reduced protein samples on the same gel, especially in adjacent lanes, since the reducing agent may have a carry-over effect on the non-reduced samples if they are in close proximity.
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We do not recommend storing reduced protein samples for long periods of time even if they are frozen because reoxidation of the sample may happen during storage, causing inconsistent results.
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*Tris-Glycine gels (except 4% Tris-Glycine gels) have a 34.5:1 Acrylamide:bisacrylamide and 2.6% Crosslinker.
*4% Tris-Glycine gels have a 76:1 ratio Acrylamide:bisacrylamide and 1.3% Crosslinker.
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The percentage of the stacking gel is 4% in most of our gels including the Bolt Bis-Tris Plus gels. The NuPAGE Tris-Acetate gels contain a 3.2% stacking gel.
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Our Invitrogen precast protein gels contain a stacking gel that is ~8 to 9 mm long (it ends right above the first ridge on the cassette). The manufacturing method used results in an interface between the stacking and resolving gels that is not visually detectable.
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*Tris-Glycine and Invitrogen Tricine Mini gels: see here (http://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf), Page 8
*NuPAGE Tris-Acetate and NuPAGE Bis-Tris Mini gels: see here (http://tools.thermofisher.com/content/sfs/manuals/nupage_tech_man.pdf), Page 10
*Bolt Bis-Tris Plus Mini gels: see here (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/bolt-bis-tris-gels.html)
*Thermo Scientific Precise Tris-HEPES gels: see here (https://tools.thermofisher.com/content/sfs/manuals/MAN0011499_Precise_Protein_Gels_UG.pdf), Page 1
*Midi gels (Invitrogen Tris-Glycine, NuPAGE Bis-Tris and NuPAGE Tris-Acetate): see here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/novex_midigel_man.pdf), Page 4
*Thermo Scientific Precise Tris-Glycine gels: see here (https://tools.thermofisher.com/content/sfs/manuals/D25MAN0011814_Precise_TrisGlycine_Gels_UG.pdf), Page 1
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