Question 1

免疫印迹检测后，我的膜上有很多点。哪里出错了？

Accepted Answer

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。

Question 2

免疫印迹检测后，我得到了很多非特异性结合。你们有何建议？

Accepted Answer

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。

Question 3

免疫印迹检测后，我得到了非常高的背景。你们有何建议？

Accepted Answer

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。

Question 4

免疫印迹检测后，我的蛋白质条带无法显色为什么？

Accepted Answer

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。

Question 5

我使用你们的一种凝胶进行蛋白质样品电泳，免疫印迹检测后得到条带模糊不清。我该怎么办？

Accepted Answer

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。

Question 6

我使用SilverXpress银染试剂盒对Tricine凝胶进行染色时，发现背景要比Tris-甘氨酸凝胶稍高一些。你们能给一些建议吗？

Accepted Answer

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。

Question 7

我进行Tricine凝胶电泳时 不小心使用了Tris-甘氨酸缓冲液。会出现什么结果？为什么？

Accepted Answer

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。

Question 8

具有很多二硫键的蛋白质样品使用BME或DTT还原后，在Tricine凝胶上出现污染的假影。是样品还原不充分吗？

Accepted Answer

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。

Question 9

你们推荐哪种转膜缓冲液用于Tricine凝胶？

Accepted Answer

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）。

Question 10

Tricine凝胶推荐的样品上样体积和蛋白质上样量是多少？

Accepted Answer

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。

Question 11

尿素能否用于Tricine凝胶系统，以获得变性结果？

Accepted Answer

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。

Question 12

Tricine系统能否用于氨基酸测序？

Accepted Answer

可以。与甘氨酸不同，三甲基甘氨酸不会干扰测序试剂。

Question 13

Invitrogen Tricine凝胶是否含有三甲基甘氨酸？

Accepted Answer

不含有，三甲基甘氨酸实际上来自于电泳缓冲液。

Question 14

为什么Tricine凝胶更适用于较小的蛋白质和多肽？

Accepted Answer

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。

Question 15

Tricine凝胶中，丙烯酰胺：双丙烯酰胺的比值以及交联剂的比例分别是多少？

Accepted Answer

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%。

Question 16

Tricine凝胶是否包含浓缩胶？

Accepted Answer

Tricine凝胶包含长度约为8-9 mm的4%浓缩胶。

Question 17

Tricine凝胶是否含有SDS？

Accepted Answer

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。

Question 18

什么情况下应该使用Tricine凝胶，而不是使用Tris-甘氨酸凝胶？

Accepted Answer

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。

Question 19

Invitrogen Tricine凝胶的保质期是多久？

Accepted Answer

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。

Question 20

有些凝胶泳道中的蛋白质条带呈不规则形或波浪形，可能原因是什么？

Accepted Answer

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

Question 21

我看到样品前方有非常不平整、不均匀的染料。能否帮我排除故障？

Accepted Answer

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。

Question 22

我在所有泳道约60 kDa处，看到一个模糊的、人为造成的双重条带。凝胶染色时间越长，该条带的颜色越深。可能原因是什么？

Accepted Answer

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。

Question 23

我在每个孔中上了不同的蛋白质样品，但是在多个相邻泳道中看到相同的蛋白质条带。可能原因是什么？

Accepted Answer

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。

Question 24

我的蛋白质条带有些倾斜或扭曲。问题出在哪里？

Accepted Answer

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。

Question 25

将蛋白质样品在变性条件下电泳，结果看到了2条蛋白条带，而我预期是看到1条条带。为什么会这样？

Accepted Answer

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。

Question 26

我的凝胶看起来正在脱离凝胶盒。可能原因是什么？

Accepted Answer

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。

Question 27

我在正常的预期蛋白条带下方看到一个微弱的阴影或“幽灵”条带。可能原因是什么？

Accepted Answer

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。

Question 28

凝胶上外侧泳道的蛋白条带出现“微笑”效应。能否帮我排除故障？

Accepted Answer

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。

Question 29

蛋白质电泳后，出现哑铃或杠铃形条带。可能原因是什么？

Accepted Answer

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。

Question 30

为什么我的蛋白质凝胶上有一个样品出现了拖尾或“皱眉”形状？

Accepted Answer

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%）

Question 31

我能否将NuPAGE抗氧化剂用于NuPAGE以外的凝胶系统，如Tricine凝胶？

Accepted Answer

不能。NuPAGE抗氧化剂在其他凝胶系统的高pH环境下无效。

Question 32

对于中型凝胶转印，你们有什么建议？

Accepted Answer

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）

Question 33

NP-40是否会影响蛋白质样品的迁移？

Accepted Answer

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。

Question 34

Invitrogen蛋白质凝胶是否含有碳水化合物？是否适用于碳水化合物分析？

Accepted Answer

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。

Question 35

Invitrogen预制胶塑料凝胶塑料卡是什么材质的？

Accepted Answer

凝胶塑料卡的材料是苯乙烯共聚物。

Question 36

Invitrogen预制胶塑料凝胶塑料卡能否重复使用？

Accepted Answer

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。

Question 37

Invitrogen小型和中型凝胶规格有何区别？

Accepted Answer

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

Question 38

你们的预制蛋白质凝胶尺寸是多少？

Accepted Answer

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

Question 39

你们的预制蛋白质凝胶是否有小型和中型规格？

Accepted Answer

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。

Question 40

我想使用同一台装置跑2块胶。是否需要将电压加倍？

Accepted Answer

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。

Question 41

我能否在同一块凝胶上跑还原型和非还原型蛋白质样品？

Accepted Answer

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。

Question 42

能否将还原型蛋白质样品保存待后续使用？

Accepted Answer

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。

Question 43

Invitrogen预制胶中，丙烯酰胺：双丙烯酰胺的比值以及交联剂的比例分别是多少？

Accepted Answer

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% 。

Question 44

你们的Invitrogen预制蛋白质凝胶中，浓缩胶的百分比是多少？

Accepted Answer

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。

Question 45

你们的Invitrogen预制蛋白质凝胶是否包含浓缩胶？

Accepted Answer

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。

Question 46

Why do Invitrogen Tricine gels work better for smaller proteins and peptides?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 47

What does it mean when bands appear to be getting narrower (or "funneling") as they progress down a protein gel?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 48

What causes dumbbell- or barbell-shaped bands during protein electrophoresis?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 49

What can cause "streaking forward" or "frowning" of samples on a SDS-PAGE gel? How can the results be improved?

Accepted Answer

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%)

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 50

Will NP-40 affect the migration of the samples in the SDS-PAGE gel?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 51

Will acetonitrile in my sample affect my electrophoresis run?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 52

What is the concentration of SDS in Invitrogen gels?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 53

What is the liquid packaged with the Invitrogen gels?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 54

If a Tricine gel heats up to around 37&deg;C during a run, should any precautions be taken?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 55

What type of transfer buffer should be used with Invitrogen Tricine gels?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 56

If a Tricine gel is accidentally run with buffers used in the Tris-Glycine system, what will happen and why?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 57

What is the cause of smeary artifacts down the lanes of a Tricine gel and how can this be prevented?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Question 58

How long should I run the Novex Tricine Gels (e.g. Cat. No. EC6675BOX) and how do I recognize the running front?

Accepted Answer

You should run the gel until the phenol red tracking dye from the Novex Tricine SDS Sample Buffer (Cat. No. LC1676) reaches the bottom of the gel. Phenol red serves as an indicator of the running front as it is a very small molecule that migrates with the ion front in Tricine gels. The Coomassie from the sample buffer runs a little slower and can be 1-2 cm behind the phenol red.

Find additional tips, troubleshooting help, and resources within our Protein Gel Electrophoresis Chambers, Power Supplies, and Accessories Support Center.

Question 59

After western detection, my membrane has a lot of spots. What could have gone wrong?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 60

I am getting a lot of non-specific binding after western detection. Can you offer some tips?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 61

I am getting very high background after western detection. Can you please offer some tips?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 62

I am unable to visualize my protein bands after western detection. What is the problem?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 63

I ran my protein sample on one of your gels and the bands look non-distinct and smeary after western detection. What should I do?

Accepted Answer

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

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Question 64

I used the SilverXpress Silver staining kit to stain my Tricine gels and noticed that the background was somewhat higher than that seen on Tris-Glycine gels. Can you please offer some suggestions?

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Novex™ 10%, Tricine, 1.0 mm, Mini Protein Gel, 10-well, 10 Gels/Box - FAQs