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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)
68 product FAQs found
•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。
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。
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。
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。
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。
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。
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）。
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。
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：
- 封闭不充分或发生非特异性结合：建议尝试使用我们的WesternBreeze封闭剂/稀释液（货号WB7050）。
- 膜污染：仅使用干净的新膜。始终佩戴干净的手套并使用镊子来处理膜。
- PVDF膜本身具有较高的背景：改为使用硝化纤维素膜。
- 硝化纤维素膜未完全湿润：遵循预润湿膜的说明。
- 印迹膜显色过度：遵循推荐的显色时间，当达到可接受的信噪比时，从底物中取出印迹膜。
- 洗膜不充分：遵循推荐的洗膜次数。在一些情况下，可能有必要增加洗膜次数和时间。
- 二抗浓度较高：通过点印迹法确定最佳抗体浓度，必要时可稀释抗体。
- 一抗浓度较高：通过点印迹法确定最佳抗体浓度，必要时可稀释抗体。
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。
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。
部分蛋白质样品可能在电泳过程中再氧化，或在电泳前未完全还原。我们推荐使用新鲜的β巯基乙醇或二硫苏糖醇（DTT）制备新鲜的样品溶液。对于NuPAGE凝胶，我们推荐在电泳缓冲液中加入抗氧化剂。
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%）
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.
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.
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.
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.
There are several reasons why streaking may occur.
(1) Sample is not completely solubilized prior to application.
(2) Sample is poorly soluble in rehydration solution.
(3) Non-protein impurities in the sample can interfere with IEF, causing horizontal streaking in the final 2-D result, particularly toward the acidic side of the gel.
(4) Ionic impurities are present in sample.
(5) Ionic detergent is present in sample.
(6) Sample load is too high.
(7) Underfocusing. Focusing time was not long enough to achieve steady state focusing.
(8) Overfocusing. Extended focusing times (over 100,000 Vh) may result in electroendosmotic water and protein movement, which can produce horizontal smearing.
What should be done?
(1) Be sure that the sample is completely and stably solubilized. Note: Repeated precipitation-resolubilization cycles produce or increase horizontal streaking.
(2) Increase the concentration of the solubilizing components in the rehydration solution.
(3) Modify sample preparation to limit these contaminants or dialyze protein.
(4) Reduce salt concentration to below 10 mM by dilution or desalt the sample by dialysis. Precipitation with TCA and acetone and subsequent resuspension is another effective desalting technique that removes lipids, nucleotides and other small molecules.
Note: Specific and non-specific losses of proteins can occur with dialysis, gel chromatography, and precipitation/resuspension of samples. If the sample preparation cannot be modified, the effect of ionic impurities can be reduced by modifying the IEF protocol. Limit the voltage to 100-150 V for 2 hours, then resume a normal voltage step program. This pre-step allows the ions in the sample to move to the ends of the IPG strip.
(5) If the ionic detergent SDS is used in sample preparation, the final concentration must not exceed 0.25% after dilution into the rehydration solution. Additionally, the concentration of the non-ionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins.
(6) Extend focusing time. Load less sample.
(7) Prolong focusing time.
(8) Reduce focusing time.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis 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.
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.
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.
1) Trim excess plastic from IPG strip
2) Equilibrate IPG strip for 2 x 15 min in 5 mL of the appropriate buffer (Use NuPAGE sample buffer for NuPAGE ZOOM gels and Tris Glycine ZOOM gels). Equilibration buffer: 1X NuPAGE sample buffer, 50 mM DTT.
3) Place equilibrated IPG strip in large well of ZOOM gel. Seal the strip in place with an agarose (0.5%) overlay.
4) Load molecular weight markers in small well of ZOOM gel.
Variations: To alkylate the proteins after reducing them, prior to separation on ZOOM gels: 1) Incubate IPG strip for 15 min in equilibration buffer 2) Transfer strip to Equilibration Buffer containing 125 mM iodoacetamide and lacking reducing agent. Incubate an additional 15 min. To denature the proteins with urea after IEF: 6 M urea can be added to Equilibration Buffer, if desired.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The length of the 2D well is 6.5 cm. The IEF gel strip must be trimmed down to 5.8-5.9 cm before positioning it into the 2D well, even after it has been soaked in methanol to shrink the gel. For the 7 cm IPG strips, significant trimming would be necessary if the gels with the 2D wells are to be used. For IPG strips, the longer IPG well (found in the ZOOM gels) is recommended.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
- 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.
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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:
- Insufficient blocking or non-specific binding: We suggest trying our WesternBreeze Blocker/Diluent (Cat. No. WB7050).
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Blot is overdeveloped: Follow recommended developing time and remove blot from substrate when signal - to -noise ratio is acceptable.
- Insufficient washing ; Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentrated secondary antibody used: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
- Concentrated primary antibody used: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
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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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Here are the possible causes and solutions:
*Staining protocol modified; Refer to the manufacturer's recommendations for correct staining protocols. To achieve best results, be sure to follow all steps exactly as given in the protocol, especially for silver staining. Changes in the protocol can result in high background.
*Background staining due to ampholytes: Prior to staining the 2D gel, thoroughly wash the gel to remove ampholytes. Use ZOOM Carrier Ampholytes as they provide a clear background due to very low non-specific binding of dyes and stains.
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We do not recommend heating protein samples containing urea over 37 degrees C as elevated temperatures cause urea to hydrolyze to isocyanate, which modifies proteins by carbamylation.
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We recommend storing them at -80 degrees C. We do not recommend storing them at -20 degrees C
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The recommended ampholyte concentration in the sample rehydration buffer is 0.5%.
*If you are loading 5-50 µg of protein (pure protein or crude lysate) per ZOOM strip, use 0.5% ampholytes in the sample rehydration buffer.
*If you are loading >50 µg of protein (crude lysate or fractionated sample) per ZOOM Strip, use 0.5-2% ampholytes in the sample rehydration buffer.
Note: Higher ampholyte concentration requires longer focusing times.
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The maximum volume of the protein sample should at most be 1/6 of the final sample volume that will be added to the strip. A good general volume would be 5-10µL. 140 µL of sample diluted in Sample Rehydration buffer is used to rehydrate each ZOOM Strip for the standard rehydration time of one hour. For overnight rehydration, we recommend using 155 µL of diluted sample.
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Alkylation prevents unwanted protein modifications by alkylating cysteines to avoid mixed disulfide formation and reoxidation and this allows for crisper focusing.
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Sorry we have discontinued selling the ZOOM 2D Protein Solubilizers.
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The main advantages of performing 2D gel electrophoresis of proteins and applications used for are listed below:
Advantages:
*Simultaneous separation of hundreds to thousands of proteins
*High capacity with superior resolution
*Compatible with further analysis by MS for protein identification and sequencing
Ability to separate and analyze low-abundance proteins
Applications:
*Comparative proteomics: identifying and analyzing differences between complex mixtures of proteins
*Protein profiling, biomarker discovery
*Separation and analysis of protein variants and isoforms
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We offer the following products for the first- and second-dimension separation of proteins:
First-dimension separation:
*Vertical gels for separation of proteins based on their isoelectric point (pI) (https://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/protein-gel-electrophoresis/protein-gels/specialized-protein-separation/isoelectric-focusing.html)
*Solution-phase isoelectric focusing of proteins using ZOOM Disks (immobilized buffer disks of specific pH) to reduce sample complexity, enrich low-abundance proteins, and increase the dynamic range of detection (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/isoelectric-focusing/zoom-ief-fractionator.html)
*Mini gel system for high-throughput isoelectric focusing of proteins using ZOOM IPG (Immobilized pH Gradient) Strips
(https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/specialized-protein-gels/2d-gel-electrophoresis/zoom-ipgrunner-system.html)
Second-dimension separation:
*ZOOM gels for 2D electrophoresis: NuPAGE Bis-Tris (Cat. No. NP0330BOX) and Tris-Glycine (Cat. No. EC60261BOX) mini gels with IPG wells ( to accommodate 7 cm ZOOM strips) for separation of proteins based on their molecular weight
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2D protein gel electrophoresis is the separation of proteins in two dimensions. In the first dimension, proteins are separated by their isoelectric point (pI) using isoelectric focusing, and in the second dimension, they are separated by their mass using SDS-PAGE.
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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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A portion of the protein sample may have re-oxidized during the run, or may not have been fully reduced prior to the run. We recommend preparing fresh sample solution using fresh beta-mercaptoethanol or dithiothreitol (DTT). For NuPAGE gels, we recommend adding antioxidant to the running buffer.
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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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