NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well, 10 gels (1 box) - FAQs

View additional product information for NuPAGE™ Bis-Tris Mini Protein Gels, 4–12%, 1.0–1.5 mm - FAQs (NP0335PK2, NP0322BOX, NP0327BOX, NP0321BOX, NP0326BOX, NP0330BOX, NP0323BOX, NP0324BOX, NP0329PK2, NP0322PK2, NP0321PK2, NP0336BOX, NP0323PK2, NP0335BOX, NP0329BOX, NP0336PK2)

95 product FAQs found

我将蛋白质置于Tris-甘氨酸凝胶上在非变性条件下进行电泳。蛋白质的pI高于Tris-甘氨酸转膜缓冲液的pH。你们建议如何对蛋白质进行转印？

•将Tris-甘氨酸转膜缓冲液的pH增加至9.2，可使pl低于9.2的所有蛋白质朝阳极方向迁移。
•使用Tris-甘氨酸转膜缓冲液，并在凝胶两侧各放一张膜。碱性高于转膜缓冲液pH的蛋白质，将被凝胶阴极侧的膜捕获。随后，可以用相同的方式处理两张膜。
•转印前，将凝胶置于含0.1% SDS的Tris-甘氨酸转膜缓冲液中孵育15分钟。少量的SDS会给予蛋白质足够的电荷，使蛋白质朝阳极端单向移动，并且在大部分情况下不会使蛋白质变性。然后，使用常规Tris-甘氨酸转膜缓冲液进行转印。

配制20X NuPAGE转膜缓冲液时，是否需要加入氯丁醇？

氯丁醇可用作NuPAGE转膜缓冲液的防腐剂，但对于蛋白质的有效转印并不是必要的。若配制缓冲液时未加入氯丁醇，则应注意缓冲液不能长期稳定保存。建议在配制后2周内用完。

NuPAGE凝胶转印能否使用碳酸盐或CAPS转膜缓冲液？

我们不建议使用碳酸盐或CAPS转膜缓冲液进行NuPAGE凝胶转印，因为这会降低转印效率。此外，这些缓冲液的高pH环境（>pH 9）会使NuPAGE抗氧化剂丧失功能。

对于改善NuPAGE凝胶上大分子蛋白的转印效果，你们有何建议？

为提高NuPAGE凝胶上大分子蛋白[高分子量蛋白]的转印效率，我们建议在安装“三明治”前，将凝胶置于含有0.02–0.04% SDS的2x NuPAGE转膜缓冲液（无甲醇）中预平衡10分钟，然后使用含甲醇和0.01%SDS的1XNuPAGE转膜缓冲液进行转印。

我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）。

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。

我已经安装了NuPAGE凝胶进行电泳并打开了电源，但是凝胶无法开始电泳，电源上也没有电压或电流读数。问题出在哪里？

•再次检查凝胶底部的胶带是否移除。
•确保凝胶安装方向正确，即凝胶盒较高的一面（有印刷字体）面向电泳装置的外侧。
•确保在缓冲液槽内部加入了足够的缓冲液，可浸没上样孔。若未浸没上样孔，检查是否有泄漏并重新封装。
•再次检查电极或Mini cell装置的连接处是否有松动。
•检查电源。

我在使用XCell SureLock Mini Cell进行NuPAGE凝胶电泳时，发现导线不适用于我的电源。你们有何建议？

您可购买ZOOM转接头，货号ZA10001，帮助您将导线连接到电源上。

在向NuPAGE凝胶上样时，看不到上样孔。你们有什么窍门吗？

我们建议在安装上样缓冲区之前，使用记号笔在凝胶盒上标记出孔的底部。此外，我们也推荐将光源正确放置在XCell SureLock装置后方，照亮实验台区域。

我在使用XCell SureLock Mini Cell进行NuPAGE凝胶电泳时，凝胶电泳时间长于正常时间。可能原因是什么？

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. 上样缓冲区泄漏：确保凝胶盒夹安置稳固，垫片在原位，且凝胶盒夹已锁定。
3. 电压设置过低：设置正确的电压。

我在从NuPAGE凝胶上转印大分子量蛋白质时出现问题。能否帮我排除故障？

对于大于100 kDa的蛋白质，我们建议在安装“三明治”前，将凝胶置于含有0.02–0.04% SDS的2X转膜液（无甲醇）中预平衡10分钟，然后使用含甲醇和0.01%SDS的1X转膜液进行转膜。

能否使用β-巯基乙醇代替NuPAGE样品还原剂？

尽管我们推荐使用NuPAGE样品还原剂以加强稳定性，但新鲜的β-巯基乙醇可以代替NuPAGE样品还原剂并获得相同的结果。通常，终浓度为2–5%的β-巯基乙醇足以还原样品。

NuPAGE Bis-Tris凝胶和NuPAGE Tris-Acetate凝胶转膜时，你们推荐在NuPAGE转膜缓冲液中加多少甲醇？

在1块胶和2块胶转膜时，建议在NuPAGE转膜缓冲液中分别加入10%和20%甲醇。

将蛋白质从NuPAGE Bis-Tris或NuPAGE Tris-Acetate凝胶上转印时，你们是否推荐在NuPAGE转膜缓冲液中加入NuPAGE抗氧化剂？

是的，我们推荐在NuPAGE转膜缓冲液中加入NuPAGE抗氧化剂，从而改善还原型蛋白质的转印结果，以便在电泳过程中维持蛋白质的还原状态。

你们推荐NuPAGE凝胶使用哪种染料？

NuPAGE凝胶可兼容所有标准考马斯染色步骤。可通过加热来加速实验，因为加热可帮助“固定”蛋白质，特别是较小的肽。NuPAGE凝胶也可兼容大部分银染操作。还能兼容铜染或锌染，以及荧光染色。

我能否一起使用NuPAGE Bis-Tris凝胶和无SDS的NuPAGE MOPS或MES上样缓冲液，在非变性条件下电泳？

我们不推荐一起使用NuPAGE Bis-Tris凝胶和无SDS的NuPAGE MOPS或MES上样缓冲液在非变性条件下电泳。这种缓冲液系统会产生过多热量，导致条带分辨率较差。此外，在中性pH环境下，不带电荷的目标蛋白可能不会很好地迁移。

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 >

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

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.

How are Bolt gels different than NuPAGE gels?

While they are both Bis-Tris based gels, the chemistries are very different since Bolt gels are optimized for western blotting. Another key difference is the wedge well design of the Bolt gels, which allows larger sample volumes to be loaded.

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

What is the advantage of NuPAGE Gels over regular Tris-Glycine gels?

The neutral operating pH of the NuPAGE Gels and buffers provides following advantages over the Laemmli system:
-Longer shelf life of 8-12 months due to improved gel stability
-Improved protein stability during electrophoresis at neutral pH resulting in sharper band resolution and accurate results (Moos et al, 1998)
-Complete reduction of disulfides under mild heating conditions (70 degrees C for 10 min) and absence of cleavage of asp-pro bonds using the NuPAGE LDS Sample buffer (pH > 7.0 at 70 degrees C)
-Reduced state of the proteins maintained during electrophoresis and blotting of the proteins by the NuPAGE Antioxidant
Please refer to the following paper: Moos M Jr, Nguyen NY, Liu TY (1988) Reproducible High Yield Sequencing of Proteins Electrophoretically Separated and Transferred to an Inert Support. J Biol Chem 263:6005-6008.

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

What is the maximum sample volume and concentration that may be loaded into the Invitrogen precast gel wells?

1-well, 1 mm: 700 µL (no more than 12 µg protein or 2.4 µg DNA per band)

2D-well, 1 mm: 400 µL or 1.5 mm: 600 µµL (no more than 12 µg protein or 2.0 µg DNA per band)

5-well, 1 mm: 60 µL (no more than 2 µg protein or 400 ng µg DNA per band)

9-well, 1 mm: 28 µL (no more than 0.5 µg protein or 100 ng DNA per band)

10-well, 1 mm: 25 µL or 1.5 mm: 37 µL (no more than 0.5 µg protein or 100 ng DNA per band)

12-well, 1 mm: 20 µL (no more than 0.5 µg protein or 100 ng DNA per band)

15-well, 1 mm: 15 µL or 1.5 mm: 25 µL (no more than 0.5 µg protein or 100 ng DNA per band)

17-well 1 mm: 15 µL (no more than 0.5 µg protein per band)

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

What percentage of stacking gel is in Invitrogen Tris-Glycine and NuPAGE Invitrogen gels? What is the length of stacking gel?

The stacking gel acrylamide concentration for Tris-Glycine and NuPAGE Bis-Tris gels is 4%. For NuPAGE Tris-Acetate gels it is 3.2%.

The stacking portion is approximately 8 to 9 mm long (it ends right above the first ridge on the cassette) on most Invitrogen protein gels (i.e., Tris-Glycine and NuPAGE gels).

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

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

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.

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

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.

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

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.

Will acetonitrile in my sample affect my electrophoresis run?

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.

What is the concentration of SDS in Invitrogen gels?

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.

Is it possible to run reduced and non-reduced samples on the same NuPAGE gel? Should the Antioxidant be used?

If the Antioxidant is omitted from the running buffer, it is possible to resolve reduced and non-reduced samples on the same gel, although the resolution may be lower. Furthermore, it is not recommended that the reduced and non-reduced samples be run side-by-side in adjacent lanes.
However, because of the neutral pH of the NuPAGE gels, the reducing agent (beta-mercaptoethanol or DTT) will not migrate through the gel with the protein the way it does in the basic environment of the Tris-Glycine gels. Instead, the reducing agent tends to remain at the top of the gel. For this reason, the NuPAGE Antioxidant is incorporated into the buffer in the upper buffer chamber. The antioxidant is able to migrate fully with the proteins and keep them reduced. As a result, it is possible that proteins prepared as non-reduced samples could become somewhat reduced during the electrophoresis run. This would result in smearing of the samples.

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

Can I use CTAB rather than SDS in my sample buffer?

No, CTAB will not work with any of our gels except for the NuPAGE Tris-Acetate gels. To use CTAB, you would need to use a running buffer of 50 mM acetic acid and 50 mM beta-alanine in equal concentrations. You would also need to switch the electrodes. Since CTAB is a cationic detergent, this would establish conditions for running a basic protein towards the anode (into the gel).

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

Do you have a protocol for extracting proteins from polyacrylamide gels?

We have a protocol for extracting proteins from polyacrylamide gels that provides a couple of options for identifying and excising the band of interest and eluting protein from gel matrix. Additionally, the article Extraction of Proteins from gels-a brief review by Kurien and Scofield (2020) provides an overview of elution procedures.

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

Do you have recommended protocols for copper or zinc staining of NuPAGE gels?

Copper or zinc staining is a rapid, sensitive method for detection of protein bands . ~10 ng reduced BSA on NuPAGE Bis-Tris gels can be detected with both the copper and zinc stain.

Copper Stain: Staining solution - 0.3 M CuCl2
After electrophoresis, remove the gel from the cassette and equilibrate the gel in 100 mL of 1X running buffer* for 15 minutes. Immerse the gel in 100 ml of 0.3 M CuCl2 solution for about 5 minutes (the protein band will appear as a negative stain with a blue background).

Zinc stain: Staining solution - 0.2 M Imidazole and 10 mM ZnCl2
After electrophoresis, remove the gel from the cassette and equilibrate the gel in 100 mL of 1X running buffer* for 15 minutes. Place the gel in 100 ml of 0.2M Imidazole solution for 10 minutes. Next immerse the gel in 100 ml of 10 mM ZnCl2 solution for about 5 minutes (the protein will appear as a transparent band with a white background).

*The 1X running buffer can be the buffer from the electrophoresis tank after run (MES, MOPS). However, for better contrast of the band, the 1X Tris-Glycine SDS running buffer is recommended.

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

Is it okay to use protein gels past their expiration date?

We do not recommend using gels past their expiration date because over time, the polyacrylamide hydrolyzes to acrylic acid and ammonia and this will affect the resolution of the proteins. Breakdown of polyacrylamide matrix is identified by:
- Ghost bands and doublets, seen first in the high molecular weight proteins
- Smiling of dye front across the gel, with bands in outer lanes becoming very slanted - proteins run slower there due to change in pH and pore size over time.


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

Can I run your protein gels overnight?

This is not really recommended, but it is always possible to increase run time by lowering the voltage of the run. In general, the relationships are linear - i.e., decreasing voltage by half will double the run time.

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

Do I need to increase the voltage when I run a 1.5 mm protein gel versus a 1.0 mm gel?

If you are running the gel at constant voltage, you do not need to increase the voltage regardless of the thickness of the gel.

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

Why do you recommend running your protein gels at constant voltage?

Using constant voltage allows the current and power to decrease during the run, providing a safety margin in case of a break in the system. Having lower power is a safety benefit and will also decrease the chances of experiencing an overheating of the gel. Further, the constant voltage setting does not need adjustment to account for differences in number or thickness of gels being run.

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

I ran my protein under native conditions on a Tris-Glycine gel. It has a pI that is higher than the pH of the Tris-Glycine transfer buffer. Can you offer some tips for transferring it?

- Increase the pH of Tris-Glycine transfer buffer to 9.2, allowing all the proteins below pI 9.2 to transfer towards the anode electrode.
- Use the Tris-Glycine transfer buffer and place a membrane on both sides of the gel. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the extra membrane placed on the cathode side of the gel. Both membranes can then be developed in the same manner.
- Prior to blotting, incubate the gel for 15 minutes in Tris-Glycine transfer buffer containing 0.1% SDS. The small amount of SDS will give the proteins enough charge to move unidirectionally towards the anode and in most cases, should not denature the protein. Proceed with the transfer using regular Tris-Glycine transfer buffer.

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

Do I need to add the chlorobutanol when making the 20X NuPAGE Transfer Buffer?

Chlorobutanol is used as a preservative in the NuPAGE transfer buffer and is not necessary for efficient transfer of proteins. You may prepare the buffer without chlorobutanol but keep in mind that the buffer will not be stable for long periods. We recommend using it within 2 weeks.

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

Can I transfer NuPAGE gels using Carbonate or CAPS transfer buffers?

We do not recommend using Carbonate or CAPS transfer buffers to transfer NuPAGE gels as the transfer efficiency will be badly compromised. Further, the high pH environment (>pH 9) of these buffers will make the NuPAGE Antioxidant non-functional.

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

Do you have any tips to improve transfer of high molecular weight proteins from NuPAGE gels?

To increase efficiency of transfer of high molecular weight proteins from NuPAGE gels, we recommend pre-equilibrating the gel in 2x NuPAGE Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x NuPAGE transfer buffer containing methanol and 0.01% SDS.

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.

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.

I have set up my NuPAGE gel to run and have switched on the power supply, but my gel is not running and there is no voltage or current reading on the power supply. What is wrong?

*Double check that the tape on the bottom of the gel has been removed.
*Make sure that the gel(s) are oriented so that the taller sides of the cassette (with the printing) are facing the outside of the electrophoresis unit.
*Make sure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer. If the wells are not covered, check for leaks and reseal.
*Double check to see if there are any loose electrodes or connections on the Mini cell unit.
*Check the power supply unit.

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

I am running my NuPAGE gel using the XCell SureLock Mini Cell but the leads do not fit into my power supply. Can you please help?

You may purchase the ZOOM adapters, Cat. No. ZA10001 to help you connect your leads to the power supply.

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

I am trying to load my samples on a NuPAGE gel but am not able to see the sample wells. Can you please suggest some tips?

We recommend marking the cassette at the bottom of the wells with a marker pen prior to assembling the upper buffer chamber. Also, we recommend illuminating the bench area with a light source placed directly behind the XCell SureLock unit.

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

I am running my NuPAGE gel using the XCell SureLock Mini Cell, and the gel run is taking longer than usual. What could be causing this?

Here are possible causes and solutions:

1) Buffers are too dilute: Check buffer recipe; remake if necessary.
2) Upper buffer chamber is leaking: Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.
3) Voltage is set too low: Set correct voltage.

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

I had problems transferring my larger molecular weight proteins from my NuPAGE gel. Can you please offer some suggestions?

For proteins larger than 100 kDa, we recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing methanol and 0.01% SDS.

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

Can I use beta-mercaptoethanol instead of the NuPAGE Sample Reducing agent?

Although we recommend using the NuPAGE Sample Reducing agent for stability reasons, fresh, neat beta-mercaptoethanol can be substituted for the NuPAGE Sample Reducing Agent, with equivalent results. A final concentration of 2-5% beta-mercaptoethanol is usually sufficient to reduce the sample.

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

How much methanol do you recommend adding to the NuPAGE transfer buffer for transfer of NuPAGE Bis-Tris gels and NuPAGE Tris-Acetate gels?

We recommend adding 10% methanol to the NuPAGE transfer buffer for transfer of one gel and 20% methanol for the transfer of 2 gels.

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

Do you recommend adding the NuPAGE Antioxidant to the NuPAGE transfer buffer when I transfer proteins from NuPAGE Bis-Tris or NuPAGE Tris-Acetate gels?

Yes, we recommend adding the NuPAGE Antioxidant to the NuPAGE transfer buffer for enhanced blotting results with reduced proteins in order to maintain the reduced state of the proteins throughout the run.

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

What stains do you recommend for NuPAGE gels?

NuPAGE gels are compatible with any of the standard Coomassie staining procedures. The protocols that are accelerated by heat are preferable as the heat serves as a “fix” for proteins, especially smaller peptides. NuPAGE gels are also compatible with most silver staining protocols. They are also compatible with copper or zinc staining, and fluorescent stains.

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

Can I use NuPAGE Bis-Tris gels with NuPAGE MOPS or MES Running Buffer prepared without SDS for electrophoresis under native conditions?

We do not recommend using NuPAGE Bis-Tris gels with NuPAGE MOPS or MES Running Buffer prepared without SDS for electrophoresis under native conditions. This buffer system may generate excessive heat, resulting in poor band resolution. Further, the protein of interest may not migrate very well in a neutral pH environment if it is not charged.

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.

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.

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.

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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.

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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.

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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.