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查看更多产品信息 NativePAGE™ Bis-Tris Mini Protein Gels, 3 to 12%, 1.0 mm - FAQs (BN1001BOX, BN1003BOX)
69 个常见问题解答
在NativePAGE电泳期间，电流下降至低于1 mA很常见。大部分电源会将此记录为“空载”错误并自动关闭，导致电泳停止。一些电源可通过禁用或关闭“负载检测”功能来避免上述情况的发生。
您可尝试采用标准的清洁建议，即先用温和的洗涤剂清洗装置，再用去离子水冲洗。
以下是可能原因和解决方案：
膜的转印垫不干净或污染。使用转印垫前，用去污剂浸泡，然后用纯水彻底清洗。转印垫破损或变色后，则更换新的转印垫。
封闭不均匀。孵育皿必须足够大，使封闭液能够完全覆盖膜。每一步都需要摇动或搅动。
以下是可能原因和解决方案：
- 膜被指纹或角蛋白污染：始终佩戴干净的手套并使用镊子来处理膜。处理膜时，仅触碰膜的边缘。
- 二抗浓度较高：按照推荐方法稀释二抗。如果背景仍然很高，但条带强度也很高，则应降低二抗的浓度。
- 一抗浓度较高：降低一抗浓度。
-一抗对蛋白标准品具有亲和力：向蛋白标准品生产商咨询蛋白标准品与一抗的同源性。
- SDS残留或转印后蛋白质与膜的结合较弱：遵循免疫检测前的膜准备说明。
- 封闭时间过短或洗膜时间过长：应确保每一步都达到指定时间。
以下是可能原因和解决方案：
- 一抗和二抗不匹配：所用二抗应该是针对一抗物种来源的抗体。
- 一抗稀释过度：1) 使用浓度更高的抗体溶液。2) 在4℃孵育更长时间（如，过夜）。3) 使用新鲜的抗体，应注意抗体溶液每使用一次，其有效抗体浓度都会有所降低。
- 封闭液含有可干扰一抗和/或二抗结合的物质：尝试交替使用封闭液± 温和的表面活性剂，如Tween-20（0.01–0.05% v/v）。基于脱脂奶粉、BSA、普通血清、明胶和这些物质的混合物以及其他原料，有多种封闭液配方可用。应注意BSA（1–5%）被认为是硝化纤维素膜的最佳封闭剂。通过点印迹法可轻松检验不同封闭液的效能。
- 一抗不能识别测试物种的相关蛋白质：1) 首先通过点印迹法评估一抗与蛋白质的反应能力。2)检查免疫原序列（如果已提供），确定您的蛋白质中是否含有该序列。3)如果无可用的免疫原序列，则通过PubMed/BLAST比对来评估您的目标蛋白与抗体的目标蛋白之间的同源程度。应注意，许多抗人源蛋白的抗体，也可识别非人源的灵长类动物蛋白，因为人源蛋白与灵长类动物蛋白具有高度的氨基酸同源性。相比之下，许多抗人源蛋白的抗体却不能识别啮齿类动物的相应蛋白（反之亦然）。应记住（注意），序列间显著的同源性并不能保证抗体可识别您的蛋白质。4)尽量每次电泳都使用推荐的阳性对照。
- 与膜结合的蛋白质不足，或样品中的目标蛋白不足：1) 凝胶上每个泳道中的蛋白质上样量至少为20–30 μg（作为起始点），因为，含量低于总蛋白量约0.2%的蛋白质，难以在免疫印迹中被检测到。2) 采用富集步骤以增加目标蛋白的浓度。例如，在转印核蛋白前制备2份细胞核裂解物，或在SDS-PAGE前进行免疫沉淀（IP）。3)减少用于裂解细胞或组织的细胞提取缓冲液的体积。4) 如果需要，应确保蛋白质提取缓冲液中使用新鲜配制的蛋白酶抑制剂和磷酸酶抑制剂。5)尽量使用推荐的阳性对照进行电泳。
- 很少或没有蛋白质转印到膜上：1) 使用可逆性蛋白质染料（如，Invitrogen可逆性膜蛋白质染料、丽春红S、酰胺黑）或预染分子量标准品，检验蛋白质转印效率。2) 确认是否使用了正确的电源极性进行转印。3)应记住，碱性pl值的蛋白质（如，组蛋白）和高分子量蛋白质的转印效果较差。4) 应记住，如果您的目标蛋白分子量较低（≤10 kDa），则转印速度可能比预期更快。5)如果您使用的是PVDF膜，应先将膜在甲醇中预浸泡，然后再浸泡在转膜液中。应注意，转膜液中的甲醇可增加膜与硝化纤维素膜的结合，但减少甲醇可增强高分子量蛋白质的转印效率。6) 低分子量蛋白质可能会穿过孔径为0.45 μm的硝化纤维素膜，因此，应改为使用0.2或0.1μm孔径的NC膜。
- 洗膜或封闭过度：1) 避免过度洗膜。若您的印迹存在其他问题，过度洗膜将导致目标蛋白无法显色。2) 避免由高浓度封闭液成分或较长孵育时间造成的过度封闭。封闭过度可妨碍抗体与蛋白的结合。明胶特别容易遮盖印迹膜上的蛋白质，因此应尽量避免使用。牛奶也会遮盖蛋白质，因此，可尝试使用0.5%牛奶或完全去除牛奶来取代封闭液中的5%牛奶。3)改为使用不同的封闭剂和/或缩短封闭时间。
- 重复使用相同的一抗稀释液 每次免疫印迹都应使用新鲜稀释的抗体，因为每重复使用一次稀释后的抗体，其有效浓度都会有所降低。同时，应记住抗体稀释液的稳定性降低，可能会很快失去活性。
- 与二抗结合的酶失效：1) 每次都使用新鲜稀释的二抗结合物。稀释液中的酶（和抗体）可能会很快失活。2) 若您使用的是辣根过氧化物酶（HRP）标记抗体，则不要在缓冲液中加入叠氮化钠。3)避免高血红素浓度（来自血液污染），否则会干扰基于HRP的检测。4) 避免在含有碱性磷酸酶-抗体结合物的缓冲液中加入磷酸盐，因为磷酸盐会抑制酶活性。
- 您的比色检测或其他检测试剂太旧并且已失活：1) 每次试验均使用新鲜的酶底物。2) 不要使用颜色发生改变或超过有效期的即用型底物试剂。3)除非产品使用手册指示，否则不要稀释底物溶液。
以下是一些建议：
•应确保每个泳道的蛋白上样量均正确——蛋白上样过多可导致条带模糊。
•低比例凝胶不能良好分离条带——尝试使用更高比例的凝胶。
•这可能是由于抗体浓度过高。我们建议遵循生产商的建议进行稀释或确定最佳抗体浓度。
在NativePAGE电泳期间，电流下降至低于1mA很常见。大部分电源会将此记录为“空载”错误并自动关闭，导致电泳停止。一些电源可通过禁用或关闭“负载检测”功能来避免上述情况的发生。
尽管凝胶的主要化学成分相同，但它们是不可交换使用的。NuPAGE Bis-Tris凝胶经过优化可用于变性和还原条件，这类凝胶的中性pH使其难以用于非变性应用，因为大部分蛋白质无电荷或具有正电荷。因此，不推荐将这类凝胶用于非变性应用。此外，NativePAGE Bis-Tris凝胶的配方不同，其经过优化可用于分辨率最高的非变性电泳。两种类型凝胶的缓冲液不可交换使用。
适用，请参考使用iBlot干转系统进行NativePAGE Bis-Tris凝胶免疫印迹分析的应用指南（https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.9778.file.dat/co01743%20native%20with%20blot%20app-f.pdf）。
我们推荐选择NuPAGE转膜缓冲液用于NativePAGE凝胶转印。PVDF是推荐使用的印迹膜，具有良好的转印和检测效果。硝酸纤维素不能兼容NativePAGE凝胶转印，这是因为硝酸纤维素膜可与Coomassie G-250染料发生紧密结合，并且不能兼容用于膜脱色和固定蛋白质的含酒精溶液。
NativePAGE Invitrogen Bis-Tris凝胶可兼容大多数标准Coomassie R-250或G-250染料配方。对于NativePAGE凝胶中最敏感的Coomassie染色，推荐使用Invitrogen Colloidal Blue染色试剂盒（货号LC6025）。由于SimplyBlue Safe Stain（货号LC6060）有特殊的固定需求，因此不推荐将其用于NativePAGE凝胶。SilverQuest银染试剂盒（货号LC6070）和SilverXpress银染试剂盒（货号LC6100）均适用于NativePAGE凝胶染色。但是，为获得最佳的总体效果和更低的背景，我们建议最好使用SilverQuest试剂盒。
不能，我们大部分蛋白质标准品（包括Invitrogen Sharp预染和未染色标准品）是经过变性和还原的，在非变性凝胶上不能良好分离。对于NativePAGE电泳，我们提供了NativeMARK未染色蛋白质标准品（货号LC0725）。这是一款即用型蛋白质标记物，可在非变性凝胶电泳中用于估计蛋白质的分子量。
请注意，即使使用非变性标准品，在非变性电泳中的分子量预估也是非常不精确的，因为各蛋白质的不同电荷和结构会严重影响凝胶迁移。为获得更精确的预估，可使用SDS-PAGE分离或质谱分析。
我们推荐使用NativeMark未染色蛋白质标准品，货号LC0725。
NativePAGE凝胶已被成功用于EMSA，对2种纯化蛋白质间的相互作用进行分析。但是，我们尚未测试将NativePAGE凝胶用于EMSA以分析核酸（DNA或RNA）与蛋白质或蛋白质复合物间的相互作用。
需要使用非离子洗涤剂进行增溶的样品不能兼容传统的非变性Tris-甘氨酸PAGE，因为随着蛋白质在聚丙烯酰胺凝胶中的迁移，会将非离子洗涤剂遗留在后方。当缺少非离子洗涤剂时，蛋白质会聚集并在泳道顶部形成垂直线条。当使用蓝色非变性电泳（NativePAGE凝胶）时，Coomassie G-250可显著减少蛋白质的聚集，分离膜上的蛋白质复合物，达到Tris-甘氨酸凝胶上得不到的效果。
此外，与Tris-甘氨酸系统的工作pH（pH 9.3–9.5）相比，NativePAGE凝胶较低的工作pH（pH 7.5–7.7）有助于维持碱性pH敏感型蛋白质的非变性结构和/或活性。
NativePAGE Invitrogen Bis-Tris凝胶的分离范围较宽，覆盖全部低分子量和高分子量范围：
•NativePAGE Invitrogen 3–12% Bis-Tris凝胶可分离分子量在30–10,000 kDa范围内的蛋白质。
•NativePAGE Invitrogen 4–16% Bis-Tris Gels凝胶可分离分子量在15–10,000 kDa范围内的蛋白质。
我们推荐将其保存在4-25℃。不可冷冻。
不同上样孔规格的NativePAGE的推荐样品上样体积和蛋白质上样量见NativePAGE Invitrogen Bis-Tris凝胶系统使用手册（https://tools.thermofisher.com/content/sfs/manuals/nativepage_man.pdf）第3页。
NativePAGE凝胶包含浓缩胶。这类凝胶的浓缩胶长度约为1cm，位于凝胶顶部，这一部分的丙烯酰胺比例较低（4%）且恒定。在浓缩胶下方的分离胶中，丙烯酰胺比例增加。但是，整个凝胶的凝胶缓冲液是相同的。所以，NativePAGE凝胶的浓缩胶不同于Laemmli系统，后者的浓缩胶具有不同的pH，导致尾随离子的迁移率降低。此外，整个NativePAGE凝胶是一次性连续灌制而成，因此，其分离胶与浓缩胶之间无界限。
NativePAGE 5% G-250样品添加剂是一种Coomassie G-250的浓缩型储液，专为与含有洗涤剂（非离子型）的NativePAGE凝胶电泳样品一起使用而设计。正常情况下，非变性蛋白在凝胶上的迁移取决于凝胶缓冲液pH下的自然电荷/等电点，但是，加入Coomassie G-250会使蛋白质产生一个关联后的负电荷，即使是通常具有正电荷的高pI蛋白质也能带上负电。该添加剂能够与蛋白质非特异性结合，无需变性。NativePAGE 5% G-250样品添加剂应加入到阴极缓冲液中，也可加到样品中。
NativePAGE 5% G-250样品添加剂（货号BN2004）是一种浓缩型Coomassie G-250储液，专为与含有洗涤剂（非离子型）的NativePAGE凝胶电泳样品一起使用而设计。NativePAGE 5% G-250样品添加剂的具体成分保密。该添加剂不含洗涤剂。
这可能是由于：
•孔中有碎片
•样品含盐量高（确保盐浓度不超过50–100 mM）
•电泳缓冲液存在问题
•制胶错误
这可能是由凝胶聚合问题和错误的样品制备（最终样品稀释度低于1X）所致。请尝试使用不同批次的相同凝胶，并确保样品正确制备。
可能原因：
还原剂过多（β-巯基乙醇）
皮肤蛋白污染物（角蛋白）
解决方案：
即将上样前，在平衡缓冲液中加入碘乙酰胺，该方法已被证明能消除这种人为条带。
处理凝胶和上样时，使用新鲜的电泳溶液并戴手套。使用高度敏感的染料时，更易出现这种问题。
可能原因：
•上样错误，导致样品残留污染了相邻孔
•电泳缓冲液污染
•凝胶灌制错误：畸形孔
解决方法：
•使用凝胶上样器将样品加到孔中
•减少上样体积
•不要延迟上样
•不要延迟电泳，因为蛋白质会水平扩散；满孔与空孔相邻时，满孔会随时间推移而逐渐污染空孔。
可能原因为：
•上样孔周围的聚合较差
•样品的盐浓度较高
•凝胶界面不均匀
•凝胶安装到夹子上时，对凝胶板造成的压力过大
•凝胶加热不均匀
•凝胶中有不溶物质或整块凝胶上的孔径不一致
•电泳时有气泡
解决方法：
•采用透析、Sephadex G-25或任何其他脱盐柱或使用Amicon浓缩管去除过多的盐或其他物质。
•电泳时，使用冷却装置或降低电流。
凝胶脱离凝胶盒的原因可能是：
•过期的凝胶发生降解。
•凝胶保存方式不恰当。
•电泳期间，电流过大导致过多的热量积累。
•聚丙烯酰胺聚合不充分。
鬼带通常被认为是由于凝胶从盒中轻微脱离（lift），导致一些样品流出到其正常迁移点之外。然后它积累起来显示为微弱的第二条带。
出现“微笑”条带可能是因为凝胶中的丙烯酰胺分解，使蛋白质迁移的基质变少。我们建议您确认使用的凝胶未超过有效期。
杠铃形条带可能是由上样量过大所致。当上样量很大时，一部分样品会扩散到孔的边缘。电泳开始后样品通过浓缩胶部分，样品不完全浓缩会使扩散到孔边缘的部分样品出现轻微滞后。较大的蛋白质在低浓度丙烯酰胺的浓缩胶中迁移阻力更大，会加剧这种效应。为缓解这一问题，我们推荐浓缩蛋白质并减少上样量。这会形成“较薄的”起始区域。
以下是可能原因和解决方案：
1. 上样量太大：上样量不要过大
2. 还原剂不新鲜：上样前正确还原样品，不要使用保存在还原剂中的样品
3. 电泳过程中，蛋白质再氧化：使用NuPAGE凝胶电泳时，在电泳缓冲液中加入抗氧化剂
4. 存在高度疏水性区域，在此区域内蛋白质排斥SDS：上样时，使用2X样品缓冲液代替1X
5. 样品含盐过多：沉淀，并使用低盐缓冲液重悬
6. 样品中SDS不足：在阴极槽加SDS（尝试0.1%、0.2%、0.3%和0.4%）
我们推荐使用NativeMark非预染蛋白质标准品，货号LC0725。
The NativePAGE Invitrogen Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analyzing native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessing purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
We recommend rinsing the membrane briefly in water, air drying and store it at room temperature in a ziplock bag. Do not place nitrocellulose in the freezer because it will shatter. Unlike PVDF, nitrocellulose membranes should never be pre-wetted in alcohol as it will cause them to shrivel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, NativePAGE gels are compatible with our Mini Gel tank, however there is a small variation from the original protocol.
The following protocol are for using NativePage gels with the Mini Gel Tank depending on if you are also performing a western transfer or not:
If you are NOT performing a western transfer:
1. Prepare 250 mL of each buffer (Anode and Dark Blue Cathode) per gel
2. After inserting a loaded NativePAGE gel into the Mini Gel Tank and pulling the clamp forward, fill the front cathode buffer chamber to the Fill Line with Dark Blue Cathode Buffer (~200 mL per gel)
3. Add 220 mL of Anode Buffer to the back anode buffer chamber for that gel. Start the gel run
If you are performing a western transfer:
1. Prepare 250 mL of Dark Blue Cathode Buffer, 250 mL of Light Blue Cathode Buffer, and 500 mL of Anode Buffer per gel
2. After inserting a loaded NativePAGE gel into the Mini Gel Tank and pulling the clamp forward, fill the front cathode buffer chamber to the Fill Line with Dark Blue Cathode Buffer (~200 mL per gel
3. Add 220 mL of Anode Buffer to the back anode buffer chamber for that gel
4. Start the gel run; pause the run after the dark blue dye has run ~1/3 of the way through gel
a. Pour out the buffers from the Mini Gel Tank
b. Refill the back anode buffer chamber with 220 mL of Anode Buffer per gel
c. Fill the front cathode buffer chamber to the Fill Line with Light Blue Cathode Buffer (~200 mL per gel)
5. Resume the gel run
Find additional tips, troubleshooting help, and resources within our Protein Biology Support Centers .
We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725 for native gel electrophoresis with Tris-Glycine, NuPAGE Tris-Acetate or NativePAGE gels.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
During NativePAGE runs, it is common for the current to drop below 1 mA. Most power supplies register this as a “No Load” error and automatically shut off, resulting in the stopping of the gel run. This can be bypassed in some power supplies by disabling or turning off the “Load Check” feature.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
You can try the standard recommendation for cleaning which would be washing the unit with a mild detergent and rinsing with deionized water.
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Here are possible causes and solutions:
- Membrane blotting pads are dirty or contaminated. Soak pads with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Blocking was uneven. The incubation dish must be sufficiently big to allow thorough coverage of membrane. Shake or agitate during each step.
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Here are possible causes and solutions:
- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.
- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.
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Here are possible causes and solutions:
- The primary antibody and secondary antibody are not compatible: Use a secondary antibody that was raised against the species in which the primary antibody was raised.
- The primary antibody is too dilute: 1) Use a more concentrated antibody solution. 2) Incubate longer (e.g., overnight) at 4 degrees C. 3) Use fresh antibody and keep in mind that each time an antibody solution is used, its effective antibody concentration decreases.
- Something in your blocking buffer interferes with binding of the primary and/or secondary antibody: Try an alternate blocking buffer ± a mild surfactant like Tween-20 (0.01-0.05% v/v). There are many blocking buffer recipes available, based on non-fat dry milk, BSA, normal serum, gelatin and mixtures of these and other materials. Note that BSA (1-5%) is considered the best blocker for nitrocellulose membranes. It is easy to check the efficacy of different blocking buffers by performing dot-blots.
- The primary antibody does not recognize the protein in the species being tested: 1) Evaluate primary antibodies by dot-blotting first to how well they react with your protein. 2) Check the immunogen sequence, if provided, and determine if it is found in your protein. 3) If no immunogen sequence is available, perform a PubMed/BLAST alignment to assess the degree of homology between your target protein and the protein against which the antibody was generated. Note that many antibodies against human proteins will also recognize the non-human primate version because there is usually a high degree of amino acid identity. In contrast, many antibodies against human proteins will not recognize the corresponding proteins from rodents (and vice versa). Remember that significant homology between sequences does not guarantee that the antibody will recognize your protein. 4) Always run the recommended positive control, if available.
- Insufficient protein is bound to the membrane or the protein of interest is not abundant enough in the sample: 1) Load at least 20-30 ?g protein per lane on your gels (as a starting point), since proteins representing less than ~0.2% of the total protein are difficult to detect on western blots. 2) Use an enrichment step to increase the concentration of the target protein. For example, prepare two nuclear lysates prior to blotting nuclear proteins or perform an immunoprecipitation (IP) prior to SDS-PAGE. 3) Reduce the volume of cell extraction buffer used to lyse your cells or tissue. 4) Be sure to use freshly prepared protease inhibitors and phosphatase inhibitors, if needed, in your protein extraction buffer. 5) Run the recommended positive control, if available.
- Poor or no transfer of the proteins to the membrane 1) Check the protein transfer efficiency with a reversible protein stain like Invitrogen Reversible Membrane Protein Stain, ponceau S, amido black or use pre-stained molecular weight standards. 2) Verify that the transfer was performed with the correct electrical polarity. 3) Remember that proteins with basic pI values (e.g., histones) and high MW may not transfer well. 4) Remember that if your target protein has a low MW (≤10 kDa), it may transfer more quickly than expected. 5) If you are using PVDF membranes, make sure to pre-soak the membrane in methanol first before soaking it in transfer buffer. Note that methanol in transfer buffer increases protein binding to nitrocellulose, but omitting methanol can increase transfer efficiency of high MW proteins. 6) Low MW proteins may pass through the 0.45 µm pores in nitrocellulose membranes, so switch to NC with 0.2 or 0.1 µm pores instead.
- Excessive washing or blocking of the membrane:- 1) Avoid over-washing the membrane. Extra washing will not allow you to visualize your protein of interest if there are other problems with your blot. 2) Avoid over-blocking by using high concentrations of the blocking buffer components or long incubation times. Too much blocking can prevent your antibodies from binding to your protein. Gelatin, in particular, can mask proteins on the blot, so avoid it, if possible. Milk can also mask proteins, so instead of using 5% milk in your blocking buffer, try using it at 0.5% instead, or remove it altogether. 3) Switch to a different blocking reagent and/or block the blot for less time.
- Using the same solution of diluted primary antibody repeatedly: Use freshly-diluted antibody for each western blot because the effective concentration of a diluted antibody decreases each time it is re-used. Also, remember that dilute solutions of antibodies are less stable and may lose their activity rapidly.
- The enzyme conjugated to your secondary antibody is not working: 1) Make a fresh dilution of your secondary antibody conjugate each time you need it. Enzymes (and antibodies) may lose activity quickly in dilute solutions. 2) Omit sodium azide in buffers if you are using HRP-conjugated antibodies. 3) Avoid high heme concentrations (from blood contamination), which can interfere with HRP-based detection. 4) Avoid using phosphate in buffers with alkaline phosphatase-antibody conjugates because phosphate inhibits enzyme activity.
- Your colorimetric or other detection reagent is old and inactive: 1) Use fresh enzyme substrate for each experiment. 2) Don't use ready-to-use substrate reagents if they have changed color on their own or if they have passed their expiration date. 3) Do not dilute substrate solutions unless instructed to do so in the product manual.
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Here are some suggestions:
- Make sure that the correct amount of protein is loaded per lane; loading too much protein can cause smearing.
- Bands will not be as well resolved in low percentage gels; try using a higher percentage gel.
- This may be due to the antibody being too concentrated. We recommend following the manufacturer's recommended dilution or determining the optimal antibody concentration
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During NativePAGE electrophoresis, it is common for the current to drop below 1 mA. Most power supplies register this as a “No Load” error and automatically shut off, resulting in the stopping of the gel run. This can be bypassed in some power supplies by disabling or turning off the “Load Check” feature.
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Although the gels share some major chemical components, they are not interchangeable. NuPAGE Bis-Tris gels are optimized for denaturing and reducing conditions, and their neutral pH makes it difficult to use them for native applications as most proteins will have no charge or positive charge. Therefore, native applications with these gels are not recommended. NativePAGE Bis-Tris gels on the other hand have a different formulation that has been optimized for native electrophoresis with highest resolution. The buffers for the two types of gels should not be interchanged.
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Yes, please take a look at the Western Blotting NativePAGE Invitrogen Bis-Tris Gels Using the iBlot 7-Minute Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.18870.file.dat/native-with-iblot-app-note-v3.pdf).
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We recommend using the NuPAGE Transfer buffer for blotting of NativePAGE gels. PVDF is the recommended blotting membrane and works well in terms of transfer and detection. Nitrocellulose is not compatible with blotting of NativePAGE gels, as the nitrocellulose membrane binds the Coomassie G-250 dye very tightly and is not compatible with alcohol-containing solutions needed to destain the membrane and fix the proteins.
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NativePAGE Invitrogen Bis-Tris Gels are compatible with most of the standard Coomassie R-250 or G-250 staining formulations. The Invitrogen Colloidal Blue Staining Kit (Cat. No. LC6025) is recommended for the most sensitive Coomassie staining in NativePAGE Gels. SimplyBlue Safe Stain (Cat. No. LC6060) is not recommended for use with NativePAGE gels, due to special fixing requirements. Both the SilverQuest Silver Staining Kit (Cat. No. LC6070) and SilverXpress Silver Staining Kit (Cat. No. LC6100) are suitable for staining of NativePAGE Gels. However, for best overall results and lower background, we recommend the SilverQuest Kit as the best choice.
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No, most of our protein standards (including the Invitrogen Sharp prestained and unstained standards) are already denatured and reduced, and will not resolve well in a native gel. For NativePAGE electrophoresis, we offer the NativeMARK Unstained Protein Standard (Cat. No. LC0725). It is a ready-to-use protein marker that allows for molecular weight estimation of proteins using native gel electrophoresis.
Please note that even with a native standard, molecular weight estimation in native electrophoresis is very inaccurate, as gel migration can be significantly affected by differing charge and conformation of individual proteins. For more accurate estimations, use SDS-PAGE separation or mass spectrometry analysis.
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We recommend using the NativeMark Unstained protein standard, Cat. No. LC0725.
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NativePAGE gels have been successfully used to perform EMSA showing protein-protein interactions between two purified proteins, but we have not tested NativePAGE gels for EMSA analysis of interactions between nucleic acids (either DNA or RNA) and a protein or protein complex.
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Samples requiring non-ionic detergents for solubility are not compatible with traditional native Tris-Glycine PAGE because as the proteins migrate into the polyacrylamide gel, they leave behind the non-ionic detergents. In the absence of non-ionic detergent, the proteins aggregate and form vertical streaks at the top of the lane. When using blue native electrophoresis (NativePAGE gels), the Coomassie G-250 dramatically reduces aggregation to allow the resolution of membrane protein complexes not seen in the Tris-Glycine gel.
In addition, compared to the operative pH of the Tris-glycine system (pH 9.3-9.5), the lower operative pH of the NativePAGE gels (pH 7.5-7.7) may help to retain the native structure and/or activity of proteins sensitive to alkaline pH.
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The NativePAGE Invitrogen Bis-Tris Gels have a wide range of separation throughout the low- and high- molecular weight ranges:
*The NativePAGE Invitrogen 3-12% Bis-Tris Gels resolve proteins in the molecular weight range of 30-10,000 kDa.
*The NativePAGE Invitrogen 4-16% Bis-Tris Gels resolve proteins in the molecular weight range of 15-10,000 kDa.
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We recommend storing them at 4-25 degrees C. They should not be frozen.
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The recommended sample loading volumes and protein loading amounts for the NativePAGE well formats are listed on Page 3 of the NativePAGE Invitrogen Bis-Tris Gel System manual (http://tools.thermofisher.com/content/sfs/manuals/nativepage_man.pdf).
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Our NativePAGE gels do have a stacking gel. The stacking gel for these gels is a ~1 cm region at the top of the gel where the acrylamide percentage is low (4%) and constant. Below the stacking gel, the acrylamide percentage begins to increase in the gradient portion of the gel. However, the gel buffer is the same throughout the gel. So the stacking gel in NativePAGE gels is not the same as in the Laemmli system where the stacking gel has a different pH causing a decreased ion mobility for the trailing ions. Also the entire NativePAGE gel is cast in one continuous delivery due to which no demarcation line is seen between the resolving (or gradient) portion of the gel and the stacking gel.
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The NativePAGE 5% G-250 Sample Additive is a concentrated stock solution of Coomassie G-250 designed for use with detergent (non-ionic) containing samples prepared for NativePAGE gel electrophoresis. Native proteins normally migrate on the gel according to natural charge/pI in the pH of gel buffer, but adding the Coomassie G-250 renders an associated negative charge even to high pI proteins that would normally have a positive charge. It binds to proteins non-specifically without denaturing them. The NativePAGE 5% G-250 Sample Additive is added to the cathode buffer and, optionally, to samples as well.
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The NativePAGE 5% G-250 Sample Additive (Cat. No. BN2004) is a concentrated stock solution of Coomassie G-250 designed for use with detergent (non-ionic) containing samples prepared for NativePAGE gel electrophoresis. The exact composition of the NativePAGE 5% G-250 Sample Additive is proprietary. It does not contain a detergent.
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This could be due to:
*Debris in the well
*High salt in the sample (make sure that the salt concentration does not exceed 50-100 mM)
*Running buffer issue
*Gel casting error
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This could be due to a gel polymerization issue combined with incorrect sample preparation (final sample dilution less than 1X). Please try a different lot of the same gel and make sure that the sample is correctly prepared.
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Possible cause:
*Excess reducing agent (beta-mercaptoethanol)
*Skin protein contaminants (keratin)
Remedy:
*The addition of iodoacetamide to the equilibration buffer just before applying the sample to the gel has been shown to eliminate these artifact bands.
*Use new electrophoretic solutions and wear gloves when handling and loading the gel. This issue is more common when highly sensitive stains are used.
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Possible cause:
*Carry-over contamination of sample from one well into neighboring wells due to loading error
*Contaminated running buffer
*Gel casting error: malformed wells
Remedy:
*Use a gel loading tip to load wells
*Reduce the sample volume
*Do not delay while loading wells
*Do not delay after the run, as proteins can diffuse horizontally; a full well left next to an empty well would eventually contaminate the empty well over time.
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Possible cause:
*Poor polymerization around sample wells
*High salt concentration in sample
*Uneven gel interface
*Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly
*Uneven heating of the gel
*Insoluble material in the gel or inconsistent pore size throughout the gel
*Air bubble during the run
Remedy:
*Remove excess salt/other material by dialysis, Sephadex G-25 or any other desalting column or using an Amicon concentrator.
*Either use a cooled apparatus or reduce the current at which electrophoresis is performed.
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Gel lifting off the cassette can be caused by:
*Expired gels that are degrading
*Improper storage of gels
*Too much heat accumulating during the electrophoresis run due to excessive current
*Insufficient polymerization of the polyacrylamide
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Ghost bands are usually attributed to a slight lifting of the gel from the cassette, which results in the trickling down of some sample beyond its normal migration point. It then accumulates and appears as a faint second band.
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"Smiling" bands may be the result of the acrylamide in the gel breaking down, leaving less of a matrix for the proteins to migrate. We recommend checking to ensure that the gels have not been used past their expiration date.
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Barbell shaped bands are a result of loading too large of a sample volume. When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will incompletely stack causing a slight retardation of the portion of the sample that diffused to the sides of the wells. This effect may be intensified for larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel. To alleviate the problem, we recommend concentrating the protein and loading a smaller volume. This gives a "thinner" starting zone.
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Here are possible causes and solutions:
1) Sample overload: Do not overload samples
2) Addition of reducing agent that is not fresh: Reduce samples right before loading and do not use samples that have been stored in reducing agent
3)Re-oxidation of the protein during the run: Add antioxidant to the running buffer if you are running NuPAGE gels
4) Presence of highly hydrophobic regions where the protein can exclude SDS: Load the sample with 2X sample buffer instead of 1X sample buffer
5) Excess salt in the sample: Precipitate and reconstitute in lower salt buffer
6) Not enough SDS in the sample: Add SDS to the upper buffer chamber (try 0.1%, 0.2%, 0.3% and 0.4% SDS)
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We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725.
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