Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm, WedgeWell™ format, 10 gels (1 box) - FAQs

View additional product information for Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm, WedgeWell™ format - FAQs (NW04125BOX, NW04122BOX, NW04127BOX, NW04120BOX)

134 product FAQs found

使用小型转印模块和Bolt Bis-Tris Plus凝胶时，每次转印需要多少甲醇？

我们建议使用含10%甲醇的1x Bolt转膜缓冲液。

使用小型转印模块对Bolt Bis-Tris Plus凝胶进行转印时，你们建议使用几块海绵垫？

我们建议一开始使用2块海绵垫。如果海绵垫随着使用而逐渐变薄，可增加使用数量。

是否有适用于小型凝胶电泳槽的湿转模块？Bolt Bis-Tris Plus凝胶是否兼容其他转印方法？

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。

我的凝胶电泳速度比正常速度快，且分辨率较差。可能原因是什么？

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。
-电压、电流或功率设置过高。将电源条件降低至推荐的电泳条件。

我的凝胶无法电泳。你们能否帮我排除故障？

请务必查看电源、设备或凝胶是否存在问题。通常，切断电源或开盖查看是否有连接错误可帮助解决问题。同时，应检查凝胶盒底部的胶带是否已去除以及缓冲液核心装置是否损坏。此外，应确保电泳槽中的缓冲液足以浸没凝胶的上样孔。

小型凝胶电泳槽可用于哪些蛋白质凝胶的电泳？

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进行电泳。请注意，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）。

Bolt Bis-Tris Plus凝胶能否使用XCell SureLock Mini-Cell进行电泳？

我们的传统Bolt Bis-Tris Plus凝胶（货号BGxxxxxBOX）已于2014年12月31日起停产，该凝胶不可使用XCell SureLock Mini-Cell进行电泳，而应使用Bolt小型凝胶电泳槽（2014年12月31日起停产）。我们的新型Bolt Bis-Tris Plus小型凝胶（货号NWxxxxxBOX）可使用XCell SureLock Mini-Cell（或小型凝胶电泳槽，货号A25977）进行电泳。

XCell SureLock Mini-Cell可用于哪些蛋白质凝胶的电泳？

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小型凝胶均可使用XCell SureLock Mini-Cell进行电泳。

我应该使用哪种电泳槽进行Invitrogen小型和中型蛋白质凝胶电泳？

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。

免疫印迹检测后，我的膜上有很多点。哪里出错了？

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。

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？

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。

免疫印迹检测后，我得到了非常高的背景。你们有何建议？

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。

免疫印迹检测后，我的蛋白质条带无法显色为什么？

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。

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？

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。

我使用你们的一种蛋白质凝胶进行蛋白质样品电泳，结果看到V形的蛋白质条带。你们能否帮我排查原因？

出现V形蛋白质条带是因为样品中存在DNA。在SDS增溶步骤后，通过增加超声处理步骤来剪切DNA，可使上述问题消失或最小化。此外，使用超速离心可从样品中去除DNA。

我在使用小型凝胶电泳槽进行Bolt Bis-Tris Plus凝胶电泳时，得到了波浪形的染色条带。为什么？

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。

我的Bolt Bis-Tris Plus凝胶发生反向电泳，如果对调Bolt小型凝胶电泳槽上的导线，则能正确电泳。能否帮我排除故障？

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)）。

我使用了小型凝胶电泳槽进行Bolt Bis-Tris Plus凝胶电泳；电泳速度比正常速度快，分辨率非常低。这是为什么？

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

我在使用小型凝胶电泳槽进行Bolt Bis-Tris Plus凝胶电泳时，电源上的电流读数很低（接近0）。问题出在哪里？

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。

我正在使用Bolt Bis-Tris Plus凝胶电泳，电泳时间比正常情况长。问题出在哪里？

以下是可能原因和解决方案：

1. 缓冲液稀释过度： 检查缓冲液配方；必要时，重新配制。
2. 缓冲液槽泄漏： 确保凝胶盒夹安置稳固，垫片在原位，且凝胶盒夹已锁定。
3. 电流设置过低： 设置正确的电流。

使用Bolt Bis-Tris Plus凝胶电泳时，只有1块胶开始电泳，而另一块不能电泳。问题出在哪里？

以下是一些可能原因和解决方法：

•缓冲液从电泳槽内部泄漏到外部。泄漏最可能是由于凝胶盒移除不恰当，导致垫片没有正确安置，密封不良。我们建议用拇指向下推垫片。
•缓冲液未完全浸没上样孔。我们建议在插入凝胶盒之前，在电泳槽中加缓冲液至电极的高度。完全填满电泳槽不仅可以防止缓冲液向外泄漏，还能起到散热的作用。
•未将凝胶盒上的胶带移除。确保在将凝胶盒插入电泳槽前移除胶带。
•未将凝胶盒放到电泳槽底部就将其夹紧（这会使底部形成电流分流，导致凝胶电泳缓慢或不能电泳）。

在向Bolt Bis-Tris Plus凝胶中上样时，看不到上样孔。我该怎么办？

我们建议在将凝胶盒放入电泳槽之前，使用记号笔在凝胶盒上标记出孔的底部，从而增加上样孔与凝胶间的对比度。

我在从Bolt Bis-Tris Plus凝胶上移除梳子时，发现存在多余的丙烯酰胺。这是为什么？

由于Bolt凝胶现有的楔形孔设计，多余的聚丙烯酰胺（可能出现于梳子区域）是我们努力确保所有孔完全形成所造成的后果。将梳子竖直拉出凝胶孔，然后颠倒凝胶并轻轻将多余的凝胶擦到垃圾桶中，可去除大部分多余的物质。我们正在努力解决这个问题。

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？

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

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？

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。

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？

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。

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？

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。

我的蛋白质条带有些倾斜或扭曲。问题出在哪里？

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。

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？

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。

我的凝胶看起来正在脱离凝胶盒。可能原因是什么？

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。

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？

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。

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？

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。

蛋白质电泳后，出现哑铃或杠铃形条带。可能原因是什么？

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。

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？

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%）

Bolt Bis-Tris Plus凝胶与NuPAGE Bis-Tris凝胶有何区别？

尽管它们都是Bis-Tris凝胶，但化学成分却相当不同。Bolt Bis-Tris Plus凝胶专为优化蛋白质免疫印迹性能而设计。另一个关键区别在于，Bolt Bis-Tris Plus凝胶的楔形孔设计使上样量增加。

NuPAGE凝胶比Invitrogen Tris-甘氨酸凝胶具有哪些主要优势？

NuPAGE凝胶比Invitrogen Tris-甘氨酸凝胶具有以下优势：

•稳定性更高，保质期更长：
- NuPAGE Bis-Tris凝胶和NuPAGE Tris-Acetate凝胶的操作pH（NuPAGE Bis-Tris凝胶为pH 7，NuPAGE Tris-Acetate凝胶为pH 8.1）比Invitrogen Tris-甘氨酸凝胶（pH 9.5）更低。在碱性pH下，聚丙烯酰胺水解为聚丙烯酸和氨，而中性pH可减少这种水解的发生。因此，NuPAGE凝胶比Invitrogen Tris-甘氨酸凝胶的稳定性更高，保质期更长（NuPAGE Bis-Tris凝胶可在4-25℃保存12个月，NuPAGE Tris-Acetate凝胶可在4℃保存8个月，而Tris-甘氨酸凝胶在4℃只能保存4-8周）。

•蛋白质分辨率更高，因为：
- 意外的化学修饰减少：在碱性pH（8.5-9.0）下，游离的丙烯酰胺可使蛋白质烷基化。其靶点是位于N端和赖氨酸上的氨基和半胱氨酸上的巯基。当pH低于8时，不会发生这种修饰。因此，与Tris-甘氨酸凝胶电泳相比，蛋白质在NuPAGE凝胶上电泳将更少发生这类意外的化学修饰。
- 蛋白质水解减少：加热Tris-甘氨酸样品缓冲液（pH 6.8），可使pH大幅降低，导致蛋白质的Asp-Pro断裂。高温和长时间加热/煮沸会增加这种断裂的发生率，导致出现多条较弱的带。在100℃时，pH降低至4.3。与其不同，NuPAGE LDS样品缓冲液（pH 8.5）加热至70℃时，pH降低至8.1，可避免发生这种断裂。

•电泳时间更短（NuPAGE Bis-Tris凝胶仅需35–50分钟，NuPAGE Tris-Acetate凝胶仅需1小时，而Tris甘氨酸凝胶则需90分钟）

Tris-甘氨酸凝胶的操作pH与NuPAGE Bis-Tris和NuPAGE Tris-Acetate凝胶有何不同？

Tris-甘氨酸凝胶的操作pH为9.5；NuPAGE Bis-Tris凝胶的操作pH为7，NuPAGE Tris-Acetate凝胶的操作pH为8.1。 < / p >

哪种预染蛋白质标准品与Bolt Bis-Tris Plus凝胶兼容？

SeeBlue预染标准品、SeeBlue Plus 2预染标准品和Invitrogen Sharp预染标准品可与与Bolt Bis-Tris Plus凝胶兼容。它们的迁移与在NuPAGE凝胶中（使用相应的缓冲液）相同。

Bolt Bis-Tris Plus凝胶是否有更小的包装（<10块凝胶/箱）？

没有，我们只提供一种规格，即10块胶/盒。

在Bolt Bis-Tris Plus凝胶上跑还原型蛋白质样品时，能否使用NuPAGE抗氧化剂？

在Bolt Bis-Tris Plus凝胶上跑还原型蛋白质样品时，没有必要使用抗氧化剂，但使用抗氧化剂也不会造成损害。

老款和新型Bolt Bis-Tris Plus凝胶有何区别？

主要区别在于凝胶尺寸不同。新型Bolt Bis-Tris Plus凝胶（货号NWxxxxxBOX）尺寸如下：
•凝胶塑料卡：10 cm x 10 cm
•凝胶：8 cm x 8 cm

新型Bolt Bis-Tris Plus凝胶电泳可使用新型小型凝胶电泳槽（货号A25977）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），则有必要升级 电泳槽，可将黑色10.5 cm凝胶凸轮型夹更换为灰色10 cm凝胶凸轮型夹（货号A26732）(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)。凸轮夹更换说明见使用手册(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)第20页或视频(https://www.youtube.com/watch?v=1FtiX8Skllw)。

老款Bolt Bis-Tris Plus凝胶（货号BGxxxxxBOX，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

老款Bolt Bis-Tris Plus小型凝胶电泳只能使用Bolt小型凝胶电泳槽（2014年12月31日起停产，将在库存售尽后停止供应）。

Bolt Bis-Tris Plus凝胶的尺寸是多少？

新型Bolt Bis-Tris Plus凝胶（货号NWxxxxxBOX）尺寸如下：
•凝胶塑料卡：10 cm x 10 cm
•凝胶：8 cm x 8 cm

老款Bolt Bis-Tris Plus凝胶（货号BGxxxxxBOX，2014年12月31日期停产）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

Bolt Bis-Tris Plus凝胶的厚度是多少？

Bolt Bis-Tris Plus凝胶的厚度为1 mm。

Bolt Bis-Tris Plus凝胶中，浓缩胶的百分比是多少？

Bolt Bis-Tris Plus凝胶含4%浓缩胶。

Bolt Bis-Tris Plus凝胶的保质期是多久？

Bolt Bis-Tris Plus可在4-25°C下保存16个月。

Bolt Bis-Tris Plus凝胶应如何保存？

我们推荐将其保存于4–25°C。不可冷冻。

Bolt Bis-Tris Plus凝胶的配方是什么？

Bolt Bis-Tris Plus凝胶具有独特的丙烯酰胺/双丙烯酰胺配方，该配方基于NuPAGE Bis-Tris凝胶的化学成分但又与之不同。

我能否使用Bolt小型凝胶电泳槽跑10cm凝胶塑料卡的小型凝胶？

使用Bolt小型凝胶电泳槽跑10 cm凝胶塑料卡的小型凝胶（不将电泳槽的10.5 cm凝胶凸轮型夹更换为10 cm凝胶凸轮型夹），请参见使用手册(https://tools.thermofisher.com/content/sfs/manuals/mini_gel_tank_man.pdf)第22页的使用说明。

注意：为得到最佳结果，使用Bolt小型凝胶电泳槽跑10 cm凝胶塑料卡的小型凝胶时，应将Bolt小型凝胶电泳槽上的黑色10.5 cm凝胶凸轮型夹更换为灰色10 cm凝胶凸轮型夹（货号A26732）。凝胶凸轮型夹更换说明见使用手册(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)第20页或视频(https://www.youtube.com/watch?v=1FtiX8Skllw)。

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)，可获取更多资源。

对于中型凝胶转印，你们有什么建议？

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）

NP-40是否会影响蛋白质样品的迁移？

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。

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？

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。

Invitrogen预制胶塑料凝胶塑料卡是什么材质的？

凝胶塑料卡的材料是苯乙烯共聚物。

Invitrogen预制胶塑料凝胶塑料卡能否重复使用？

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。

Invitrogen小型和中型凝胶规格有何区别？

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

Bolt Bis-Tris Plus凝胶是否具有中型规格？

Bolt Bis-Tris Plus凝胶无中型规格。

你们的预制蛋白质凝胶尺寸是多少？

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

你们的预制蛋白质凝胶是否有小型和中型规格？

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。

我想使用同一台装置跑2块胶。是否需要将电压加倍？

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。

我能否在同一块凝胶上跑还原型和非还原型蛋白质样品？

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。

能否将还原型蛋白质样品保存待后续使用？

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。

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？

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% 。

你们的Invitrogen预制蛋白质凝胶中，浓缩胶的百分比是多少？

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。

你们的Invitrogen预制蛋白质凝胶是否包含浓缩胶？

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。

使用你们的预制蛋白质凝胶时，推荐的样品上样体积和蛋白质上样量是多少？

•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

你们的预制蛋白质凝胶是否含有SDS？

我们的预制蛋白质凝胶不含SDS，但在使用合适的变性电泳缓冲液时，可在变性条件下电泳。
注意：NuPAGE Bis-Tris凝胶、Bolt Bis-Tris Plus凝胶和Thermo Scientific Precise Tris-HEPES凝胶不可在非变性条件下电泳；这些凝胶只能用于变性条件下。

*Invitrogen Tris-甘氨酸凝胶：非变性电泳时使用Invitrogen Tris-甘氨酸非变性电泳缓冲液 。变性电泳时使用 Invitrogen Tris-甘氨酸SDS电泳缓冲液

*Invitrogen NuPAGE Tris-Acetate凝胶：非变性电泳时使用Invitrogen Tris-甘氨酸非变性电泳缓冲液 。变性电泳时使用 NuPAGE Tris-乙酸SDS电泳缓冲液

*Invitrogen NuPAGE Bis-Tris凝胶：使用NuPAGE MOPS-SDS电泳缓冲液或NuPAGE MES-SDS电泳缓冲液进行变性电泳

*Invitrogen Bolt Bis-Tris Plus凝胶：使用Bolt MOPS SDS电泳缓冲液或Bolt MES SDS电泳缓冲液进行变性电泳

*Thermo Scientific Precise Tris-甘氨酸凝胶：非变性电泳时使用Tris-甘氨酸SDS电泳缓冲液，不加入SDS。变性电泳时使用Tris-甘氨酸SDS电泳缓冲液。

*Thermo Scientific Precise Tris-HEPES凝胶：使用Tris-HEPES SDS电泳缓冲液进行变性电泳。

Can I prepare my protein sample with the reducing agent and store it for future use?

DTT is not stable, so it must be added and the reduction performed just prior to loading your samples.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

My LDS or SDS sample buffer precipitates when stored at 4 degrees C. Can I warm it up? Can I store it at room temperature?

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.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

What are the storage conditions for Bolt gels?

Similar to NuPAGE gels with storage temperatures of 4 to 25 degrees C.

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

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

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.

How much methanol is required per transfer when using the Mini Blot Module and Bolt Bis-Tris Plus gels?

We recommend using 1x Bolt Transfer buffer with 10% methanol.

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

How many sponge pads do you recommend using for transfer of Bolt Bis-Tris Plus gels using the Mini Blot Module?

We recommend using 2 sponge pads to start with. If the sponge pads become thinner with extended use, then an additional sponge pad can be used.

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

Is there a wet transfer blot module available for the Mini Gel Tank? Are Bolt Bis-Tris Plus gels compatible with other transfer methods?

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.

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

My gel run is faster than normal with poor resolution. What could be causing this problem?

Here are possible causes and solutions:

- Buffers are too concentrated or incorrect. Check buffer recipe; dilute or re-make if necessary.
- Voltage, current or wattage is set at a higher limit. Decrease power conditions to recommended running conditions.

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

My gel will not run. Can you please help me troubleshoot?

It is important to determine whether the problem is with the power supply, the apparatus or the gel. Often, it helps to switch out the power supply or the lid to see if there is a faulty contact. Also, check to see whether the tape from the bottom of the gel cassette has been removed and whether the buffer core is damaged. Additionally, make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.

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

Which of your protein gels can I run using the Mini Gel Tank?

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. Please note that 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).

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

Can I run the Bolt Bis-Tris Plus gels in the XCell SureLock Mini-Cell?

Our Original Bolt Bis-Tris Plus gels (Cat. No. BGxxxxxBOX), discontinued as of December 31, 2014) do not fit in the XCell SureLock Mini-Cell. They need to be run in the Bolt Mini Gel Tank (discontinued as of December 31, 2014). Our New Bolt Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX) can be run using the XCell SureLock Mini-Cell (or Mini Gel Tank, Cat. No. A25977).

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

Which of your protein gels can I run using the XCell SureLock Mini-Cell?

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 XCell SureLock Mini-Cell.

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

Which gel chamber should I use to run your Invitrogen Mini and Midi protein gels?

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.

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

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

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.

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

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.

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

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.

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

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.

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?

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.

I ran my protein samples on one of your protein gels and saw V-shaped protein bands. Can you please help me troubleshoot?

V-shaped protein bands are caused by the presence of DNA in the sample. The artifact might be eliminated or minimized by shearing the DNA with additional sonication after the SDS-solubilization step. Alternatively, the DNA can be removed from the sample using an ultra-centrifuge.

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

I am getting wavy dye fronts when using Bolt Bis-Tris Plus gels with the Bolt Mini Gel Tank. Why is this?

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.

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

My Bolt Bis-Tris Plus gel is running backwards but if I reverse the leads on the Bolt Mini Gel Tank, it runs correctly. Can you please help me troubleshoot?

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).

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

I ran my Bolt Bis-Tris Plus gel using the Mini Gel Tank; the run was faster than normal and the resolution was very poor. What happened?

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.

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

I am running a Bolt Bis-Tris Plus gel using the Mini Gel Tank and the current reading on the power supply is very low (close to zero). What is the problem?

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.

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

I am running a Bolt Bis-Tris Plus gel and the run is taking longer than usual. What is the problem?

Here are possible causes and solutions:

1) Buffers are too dilute: Check buffer recipe; remake if necessary.
2) Buffer chamber is leaking: Make sure the cassette clamp is firmly seated, the gaskets are in place and the cassette clamp is locked.
3) Current is set too low: Set correct current

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

I am running a Bolt Bis-Tris Plus gel, and only one gel is running while the other isn't. What is the issue?

Here are some likely reasons and remedies:

*Leaking of buffer from the inside of the tank to the outside. Leaking is most likely due to inappropriate removal of cassettes, which can lead to the gasket not sitting properly and loss of a good seal. We recommend pushing the gasket down with the thumb.
*Not covering the wells completely with the buffer. We recommend filling the tank up to the electrodes prior to inserting the gel cassettes. Complete filling of the tank not only prevent leaks from the inside to the outside but also acts as a heat sink.
*Not removing the tape from one of the gel cassettes. Make sure that the tape is removed from both gel cassettes before inserting them into the tank.
*Not placing the gel cassette all the way to the bottom of the tank prior to clamping it (this will create a bypass of the current at the bottom, so the gel will run slower or not run at all).

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

I am trying to load my Bolt Bis-Tris Plus gel but am unable to see the sample wells. What should I do?

We recommend increasing the contrast between the sample well and the rest of the gel by marking the cassette at the bottom of the wells with a marker pen prior to placing the cassette in the electrophoresis tank.

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

When I tried to remove the comb from my Bolt Bis-Tris Plus gel, I noticed the presence of extra acrylamide. What is the purpose?

Due to the current wedge-well design of our Bolt gels, excess polyacrylamide (likely to be present in the area of the comb) is a consequence of our efforts to ensure that all wells are completely formed. Most of this excess material can be removed by slowly pulling the comb straight up out of the gel wells, turning the gel upside down, and then gently flicking the excess gel into a trash receptacle. Efforts are underway to reduce this excess.

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

The protein bands in some of my gel lanes are irregular or wavy? What would have caused this problem?

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

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

I am seeing a very wavy and uneven dye front with my samples. Can you please help me troubleshoot?

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.

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

I am seeing a faint, artifact doublet band at ~60 kDa in all my lanes. This band seems to be getting darker the longer I stain the gel. What could be causing this?

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.

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

I loaded different protein samples in each well but I see the same protein band in several neighboring lanes. What could have happened?

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.

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

My protein bands appear to be skewed or distorted. What is the problem?

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.

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

I ran my reduced protein samples under denaturing conditions and am seeing doublet protein bands when I expect to see single bands. Why is this happening?

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.

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

My gel seems to be lifting off the cassette. What could be causing this?

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

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

I am seeing a faint shadow, or "ghost" band below a normal and expected protein band? What could be the potential issue?

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.

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

My protein bands in the outer lanes of the gel show a "smiling" effect. Can you please help me troubleshoot?

"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.

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

I see dumbbell or barbell shaped bands after protein electrophoresis. What could be causing this?

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.

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

Why do I get streaking forward or "frowning" of one of my samples on my protein gel?

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)

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

How are Bolt Bis-Tris Plus gels different from NuPAGE Bis-Tris gels?

While they are both Bis-Tris based gels, the chemistries are very different as Bolt Bis-Tris Plus gels have been optimized for western blotting. Another key difference is the enabling wedge well design for Bolt Bis-Tris Plus gels, which allows larger sample volume load.

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

What are the main advantages of NuPAGE gels over Invitrogen Tris-Glycine gels?

NuPAGE gels have the following advantages over Tris-Glycine gels:

*Higher stability and longer shelf life: NuPAGE Bis-Tris gels and NuPAGE Tris-Acetate gels have a lower operating pH (pH 7 for NuPAGE Bis-Tris gels and pH 8.1 for NuPAGE Tris-Acetate gels) than Invitrogen Tris-Glycine gels (pH 9.5). At basic pH, polyacrylamide hydrolyzes to polyacrylic acid and ammonia whereas at neutral pH, this hydrolysis is slower. Hence, NuPAGE gels have higher stability and longer shelf life than Invitrogen Tris-Glycine gels (12 months at 4-25 degrees C for NuPAGE Bis-Tris gels and 8 months at 4 degrees C for NuPAGE Tris-Acetate gels vs 4-8 weeks at 4 degrees C for Tris-Glycine gels).

*Better resolution of proteins due to:

- Reduced undesired chemical modifications: Free acrylamide alkylates proteins at basic pH (8.5 to 9.0). It targets sulfhydryl cysteines and amine groups at the N-terminus and on lysines. This modification does not happen at pH below 8. Hence, proteins run on NuPAGE gels undergo fewer of these undesired chemical modifications than those run on Tris-Glycine gels.

- Reduced hydrolysis of proteins: Heating of Tris-Glycine sample buffer (pH 6.8) results in a drop in pH, causing Asp-Pro cleavage of proteins. High temperature and longer duration of heating/boiling increase the rate of this cleavage resulting in multiple peptide bands of decreased intensity. At 100 degrees C, the pH drops as low as pH 4.3. On the other hand, NuPAGE LDS sample buffer (pH 8.5) drops to pH 8.1 when heated to 70 degrees C, avoiding this cleavage.

*Faster run times: 35-50 min for NuPAGE Bis-Tris gels and 1 hour for NuPAGE Tris-Acetate gels vs 90 min for Tris-Glycine gels

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

How does the operating pH for Tris-Glycine gels differ from that for NuPAGE Bis Tris and NuPAGE Tris-Acetate gels?

The operating pH for Tris-Glycine gels is 9.5; the operating pH for NuPAGE Bis-Tris gels is 7 and for NuPAGE Tris-Acetate gels is 8.1.

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

Which pre-stained protein standards are compatible with Bolt Bis-Tris Plus gels?

SeeBlue prestained standard, SeeBlue Plus 2 prestained standard and Invitrogen Sharp prestained standard are compatible with Bolt Bis-Tris Plus gels. Their migration is the same as that in NuPAGE gels with the corresponding buffer.

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

Do you offer Bolt Bis-Tris Plus gels in smaller packs (<10 gels/box)?

No. We only offer one size of 10 gels per box.

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

Should I use the NuPAGE Antioxidant when I run reduced protein samples in Bolt Bis-Tris Plus gels?

It is not necessary to use the Antioxidant while running reduced samples in Bolt Bis-Tris Plus gels but wouldn't hurt to use it.

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

What is the difference between your original and new Bolt Bis-Tris Plus gels?

The main difference is in the dimensions of the gels. Our New Bolt Bis-Tris Plus gels (Cat. No. NWxxxxxBOX) have the following dimensions:

*Cassette: 10 cm x 10 cm
*Gel: 8 cm x 8 cm

The new Bolt Bis-Tris Plus gels can be run using the new Mini Gel Tank (Cat. No. A25977), or the 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) (http://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). Instructions for replacement of the cam handles can be found on Page 20 of the manual or in this video (https://www.youtube.com/watch?v=1FtiX8Skllw).

Our Original Bolt Bis-Tris Plus gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) had slightly different dimensions:

*Cassette: 10 cm x 10.5 cm
*Gel: 8 cm x 8.3 cm

Our Original Bolt Bis-Tris Plus Mini gels can only be run in the Bolt Mini gel tank (discontinued as of December 31, 2014, and will be offered until inventory is depleted).

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

What are the dimensions of Bolt Bis-Tris Plus gels?

Our New Bolt Bis-Tris Plus gels (Cat. No. NWxxxxxBOX) have the following dimensions:

*Cassette: 10 cm x 10 cm
*Gel: 8 cm x 8 cm

Our Original Bolt Bis-Tris Plus gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) had slightly different dimensions:

*Cassette: 10 cm x 10.5 cm
*Gel: 8 cm x 8.3 cm

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

What is the thickness of Bolt Bis-Tris Plus gels?

Bolt Bis-Tris Plus gels have a 1 mm thickness.

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

What is the percentage of the stacking gel in Bolt Bis-Tris Plus gels?

Bolt Bis-Tris Plus gels contain a 4% stacking gel.

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

What is the shelf life of Bolt Bis-Tris Plus gels?

Bolt Bis-Tris Plus gels have a shelf life of 16 months when stored at 4-25 degrees C.

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

How should I store my Bolt Bis-Tris Plus gels?

We recommend storing them at 4-25 degrees C. They should not be frozen.

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

What is the formulation of Bolt Bis-Tris Plus gels?

Bolt Bis-Tris Plus gels have a proprietary acrylamide/bisacrylamide formulation that is based on the NuPAGE Bis-Tris gel chemistry but different from it.

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

Can I run Mini gels with 10 cm gel cassettes using a Bolt Mini Gel Tank?

To run Mini gels with 10 cm gel cassettes using a Bolt Mini Gel Tank (without replacement of 10.5 cm cassette clamp cam handles with 10 cm cassette clamp cam handles), please use the instructions provided on Page 22 of the manual (https://tools.thermofisher.com/content/sfs/manuals/mini_gel_tank_man.pdf).

Note: For optimal results, to run 10 cm cassette Mini gels with a Bolt Mini Gel Tank, one should replace the black 10.5 cm cassette clamp cam handles on the Bolt Mini Gel Tank with gray 10 cm cassette clamp cam handles (Cat. No. A26732). Instructions for replacement of the cam handles can be found on Page 20 of the manual (http://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) or in this video (https://www.youtube.com/watch?v=1FtiX8Skllw).

Additional resources 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).

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

How do you recommend transferring Midi gels?

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).

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

Will NP-40 affect the migration of my protein samples?

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.

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

Do your Invitrogen protein gels contain any carbohydrates and are they suitable for carbohydrate analysis?

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.

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

What is the material used for making your Invitrogen precast gel plastic cassettes?

The cassettes are made of a styrene copolymer.

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

Can I recycle your Invitrogen precast gel plastic cassettes?

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.

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

What is the difference between Invitrogen Mini and Midi gel formats?

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.

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

Do you offer Bolt Bis-Tris Plus gels in the Midi format?

Bolt Bis-Tris Plus gels are not available in the Midi format.

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

What are the dimensions of your precast protein gels?

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.

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

Are your precast protein gels available in Mini and Midi formats?

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.

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

When running two protein gels, do I need to double the voltage?

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.

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

Can I run reduced and non-reduced protein samples on the same gel?

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.

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

Can I store my reduced protein samples for later use?

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.

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

What is the ratio of acrylamide:bisacrylamide and percentage of cross-linker in your Invitrogen precast gels?

*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.

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

What is the percentage of the stacking gel in your Invitrogen precast protein gels?

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.

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

Do your Invitrogen precast protein gels contain a stacking gel?

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.

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

What are the recommended sample loading volumes and protein loading amounts for your precast protein gels?

*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

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

Do your precast protein gels contain SDS?

Our precast protein gels do not contain SDS but they can be run under denaturing conditions when used with the appropriate denaturing running buffer.
Note: NuPAGE Bis-Tris gels, Bolt Bis-Tris Plus gels, and Thermo Scientific Precise Tris-HEPES gels cannot be run under native conditions; they can only be run under denaturing conditions.

*Invitrogen Tris-Glycine gels: For Native electrophoresis, use Invitrogen Tris-Glycine Native Running Buffer. For Denaturing electrophoresis, use Invitrogen Tris-Glycine SDS Running Buffer

*NuPAGE Tris-Acetate gels: For Native electrophoresis, use Invitrogen Tris-Glycine Native Running Buffer. For Denaturing electrophoresis, use NuPAGE Tris-Acetate SDS Running Buffer

*NuPAGE Bis-Tris gels: For Denaturing electrophoresis, use NuPAGE MOPS-SDS Running Buffer or NuPAGE MES-SDS Running Buffer

*Bolt Bis-Tris Plus gels: For Denaturing electrophoresis, use Bolt MOPS SDS Running Buffer or Bolt MES SDS Running Buffer

*Thermo Scientific Precise Tris-Glycine gels: For Native electrophoresis, use Tris-Glycine SDS Running Buffer without SDS added. For Denaturing electrophoresis, use Tris-Glycine SDS Running Buffer.

*Thermo Scientific Precise Tris-HEPES gels: For Denaturing electrophoresis, use Tris-HEPES SDS Running Buffer.

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