Novex™ 4 to 20% Tris-Glycine Plus, 1.0 mm, Midi Protein Gels, 26-well, w/adapters, 10 gels (1 box) - FAQs

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78 product FAQs found

我使用Colloidal Blue染色剂对Tris-甘氨酸凝胶进行染色，发现低比例丙烯酰胺的Tris-甘氨酸凝胶比高比例丙烯酰胺的Tris-甘氨酸凝胶的背景更高。原因是什么？如何解决？

丙烯酰胺比例低于10%的凝胶具有较大的孔，胶体会渗透入孔并将其封闭，从而产生较高的背景。为降低过高的背景，可将凝胶在25%甲醇溶液中孵育，直至获得清晰的背景。应注意，这样做会部分去除条带上的染料，在>25%甲醇溶液中长时间孵育会导致蛋白质条带和背景完全脱色。

我使用了Colloidal Blue染色试剂盒对Tris-甘氨酸凝胶进行染色，结果得到了非常高的背景。你们有能够降低背景的窍门吗？

使用NuPAGE Invitrogen Bis-Tris凝胶/小肽附带的实验方案，可能会降低背景。该实验方案包含额外的固定步骤以除去多余的SDS，SDS是一种抗胶体试剂，可导致较高背景。染色液的低pH条件可固定凝胶，但没有NuPAGE实验方案特有的预固定步骤速度快。

我已经安装了Tris—甘氨酸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Tris-甘氨酸凝胶电泳时，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Tris—甘氨酸凝胶上样时，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Tris-甘氨酸凝胶电泳时，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Tris-甘氨酸凝胶上进行蛋白质电泳，样品在凝胶底部停止电泳而标记物正常电泳。为什么会出现这种情况？

若使用了含抗氧化剂NuPAGE电泳缓冲液进行Tris-甘氨酸凝胶电泳，可能出现这种情况。请确保在Tris-甘氨酸凝胶电泳时使用正确的Tris-甘氨酸电泳缓冲液。

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。

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%）

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 >

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。

在非变性条件下进行凝胶电泳，推荐使用哪种蛋白质标准品？

我们推荐使用NativeMark非预染蛋白质标准品，货号LC0725。

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 Tris-Glycine Plus Midi Gels be stored at room temperature?

No. They must be stored at 4 degrees C.

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

Do Tris-Glycine Plus Midi Gels require any specific buffers?

Tris-Glycine Plus Midi Gels can use standard Tris-Glycine sample and running buffers. If you want to run under native conditions, we recommend using sample and running buffers that do not contain SDS (or our Native Tris-Glycine premade buffers).

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What in the chemistry changed to allow for a longer shelf life for Tris-Glycine Plus Midi Gels?

We were able to optimize shelf life of the gels through a proprietary gel formulation change. Unfortunately, we are unable to provide specific details on the chemical changes.

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

What is the shelf life of Tris-Glycine Plus Midi Gels?

Tris-Glycine Plus Midi Gels have a shelf life of up to 1 year depending upon gel percentages. The minimum shelf life of these gels is at least 6 months from date of manufacture.

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

Can Tris-Glycine Plus Midi Gels be run in the Bio-Rad Criterion gel running tank?

Yes, Tris-Glycine Plus Midi Gels can be run in the Bio-Rad Criterion tank with the help of adapters. If you are interested in running these gels in the Bio-Rad Criterion tank, we recommend buying the versions of these gels that come with adapters.

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

With the launch of Tris-Glycine Plus Midi gels, will the current Tris Glycine Midi Gels be discontinued?

Yes, the current Tris-Glycine Midi Gels will be discontinued on December 31 2017.

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

I stained Tris-Glycine gels with Colloidal Blue stain and noticed that the background was higher in low acrylamide percentage Tris-Glycine gels compared to high acrylamide percentage Tris-Glycine gels. Why is this and how can I resolve it?

Background is generally higher in gels with less than 10% acrylamide percentage due to penetration and trapping of colloids within the large pores of these gels. Excess background may be reduced by incubating the gel in 25% methanol solution until a clear background is obtained. Be aware that the dye will also be partially removed from the bands and that prolonged incubation in >25% methanol will result in complete destaining of protein bands and background.

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I used the Colloidal Blue staining kit for staining my Tris-Glycine gels and got very high background. Can you please offer some tips to reduce the background?

It may be possible to reduce background by using the protocol provided for NuPAGE Invitrogen Bis-Tris gels/Small Peptides. This protocol incorporates an extra fix step to remove excess SDS, which can act as an anti-colloidal agent and lead to higher background. The low pH of the staining solution will fix the gel, but not as fast as the pre-fix step specified in the NuPAGE protocol.

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I have set up my Tris-Glycine 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 Tris-Glycine 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 Tris-Glycine 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 Tris-Glycine 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 volatage

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

I ran my protein samples on a Tris-Glycine gel and the samples stopped running in the bottom portion of the gel whereas the marker ran fine. Why did this happen?

This can happen if Tris-Glycine gels are run using NuPAGE Running buffer containing Antioxidant. Please make sure that the correct Tris-Glycine Running buffer is used with Tris-Glycine gels.

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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.

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.

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.

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.

Which protein standard do you recommend using with gels run under native conditions?

We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725.

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.