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View additional product information for Novex™ Sharp Pre-stained Protein Standard - FAQs (LC5800)
34 product FAQs found
褪色最可能是由于封闭/洗涤液中的洗涤剂将一些蛋白质从膜上洗脱造成的。染料本身不会从蛋白质上洗脱，因为它们是共价结合的。我们已经发现较小孔径的膜可以在封闭和洗涤过程中更好地保留蛋白质，因此，为了获得绝佳的分辨率和蛋白质保留率，推荐使用0.2 µm代替0.45 µm的膜。转印后，可以用铅笔在膜上圈出预染条带，从而在封闭和处理后仍可辨认条带位置。
•降低电压、电流或缩短转印时间
•确保转膜缓冲液的甲醇浓度合适；可使用浓度为10–20%的甲醇，从而去除SDS-蛋白质复合物中的SDS，并促进蛋白质与膜的结合。
•确保转膜缓冲液的SDS浓度合适（若加入了SDS），SDS浓度不要超过0.02–0.04%。过多的SDS会阻碍蛋白质与膜的结合。过多的SDS会阻碍蛋白质与膜的结合。
•检查膜的孔径和靶标蛋白质的大小。小于10kDa的蛋白质很容易穿过0.45μm孔径的膜。如果您的目标蛋白质小于10 kDa，那么最好使用0.2μm孔径的膜。
•增加电压、电流或转印时间
•凝胶和SDS-蛋白质复合物中的SDS会促进蛋白质从凝胶中洗脱，但抑制蛋白质与膜的结合。这种抑制作用在硝化纤维素膜上的强度大于PVDF膜。对于难以从凝胶中洗脱的蛋白质，如大分子量蛋白质，可在转膜缓冲液中加入少量SDS以改善转印效果。我们建议在组装三明治前将凝胶置于含0.02–0.04% SDS的2x转膜缓冲液（无甲醇）中预平衡10分钟，然后使用含10%甲醇和0.01% SDS的1X转膜缓冲液进行转印。
•甲醇可去除SDS-蛋白质复合物中的SDS，促进蛋白质与膜的结合，但对凝胶本身有一些不良影响，会降低转印效率。甲醇可能导致孔径减小、某些蛋白质发生沉淀以及一些碱性蛋白质带正电荷或变为中性。应确保转膜缓冲液的甲醇浓度不高于10–20%，并使用高质量的分析级甲醇。
预染标准品具有与每种蛋白质共价结合的染料，这将导致标准品在不同的缓冲系统（即不同的凝胶）中迁移率不同。因此，使用预染标准品进行分子量估算将仅得出蛋白质的表观分子量。预染标准品可用于分子量估算、确认凝胶迁移和估算转膜效率，但对于需要精确估算分子量的应用，应使用非预染标准品。
•上样时，请注意确保相邻样品泳道没有交叉污染。
•确保每个泳道上标准品的量都是正确的。蛋白质上样过多会导致产生额外的条带，这个问题在使用银染凝胶时尤为突出。
•标准品储存不当或反复冻融会导致蛋白质降解。
建议如下：
•确保每个泳道上标准品的量都是正确的。蛋白质上样过多会导致模糊，这个问题在使用银染凝胶时尤为突出。
•条带在低百分比凝胶中不能很好地分辨。尝试使用更高百分比的凝胶。
•如果在转膜/检测后条带看起来不明显和模糊，可能是由于抗体浓度过高。遵循制造商建议的稀释度或通过斑点印迹确定最适抗体浓度。
建议如下：
•检查使用的凝胶类型/凝胶百分比。可能会由于凝胶类型和/或百分比的不同而不能看到所有条带。例如，蛋白质标准品的最小条带可能不能在非常低百分比的凝胶上分辨，而较高分子量条带可能不能在高百分比凝胶上分辨。
•检查蛋白质分子量标准品的有效日期。由于蛋白质降解，过期批次可能导致条带褪色或缺失。
•检查蛋白质分子量标准品的储存条件。不适当的储存条件会损害标准品中蛋白质的稳定性。
•确保蛋白质分子量标准品在上样到凝胶上之前未加热/煮沸。我们的蛋白质分子量标准品可直接上样，我们不建议将其加热/煮沸，因为这可能会导致标准品中的蛋白质降解。
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。
SeeBlue预染标准品、SeeBlue Plus 2预染标准品和Invitrogen Sharp预染标准品可与与Bolt Bis-Tris Plus凝胶兼容。它们的迁移与在NuPAGE凝胶中（使用相应的缓冲液）相同。
The 3.5 kDa band is visible only on NuPAGE gels with 1X MES SDS running buffer.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Invitrogen Sharp Pre-Stained Standard molecular weight estimations are the same in NuPAGE Bis-Tris, NuPAGE Tris-Acetate, Invitrogen Tris-Glycine, and Tricine Gels. The appearance/migration of the protein bands may differ depending upon the gel type and running conditions
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Invitrogen protein standards do not require any additional preparation. Thaw protein standards at room temperature, vortex to mix well, and load.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The fading is most likely due to detergent in the western blocking/washing solutions that can remove some of the proteins from the membrane. The dye itself will not wash off of the proteins because it is covalently bound. We have found that smaller pore size membranes retain the proteins better during blocking and wash procedures, and hence recommend use of 0.2 µm instead of 0.45 µm membranes for best resolution and protein retention. After transfer, it is a good idea to circle the pre-stained bands with a pencil on the membrane, so band positions can be identified after blocking and processing.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
- Decrease voltage, current or length of transfer time
- Make sure that the methanol concentration in the transfer buffer is proper; use a methanol concentration of 10-20% methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane.
- Make sure that the SDS concentration (if added) in the transfer buffer is proper, don't use more than 0.02-0.04% SDS. Using too much SDS can prevent binding of proteins to the membrane.
- Check the pore size of the membrane and the size of the target protein. Proteins smaller than 10 kDa will easily pass through a 0.45 µm pore size membrane. If proteins smaller than 10 kDa are of interest, it would be better to use a 0.2 µm pore size membrane.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
- Increase voltage, current or length of time for transfer
- SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes. This inhibition is higher for nitrocellulose than for PVDF. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. We recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing 10% methanol and 0.01%SDS.
- Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Make sure that the methanol concentration in the transfer buffer is not more than 10-20% and that high-quality, analytical grade methanol is used.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Pre-stained standards have a dye that is covalently bound to each protein that will result in the standard migrating differently in different buffer systems (i.e., different gels). As a result, using a pre-stained standard for molecular weight estimation will only give the apparent molecular weight of the protein. Pre-stained standards may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained standard should be used.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
- While loading, take care to make sure that there is no cross-contamination from adjacent sample lanes.
- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can result in extra bands and this is a problem especially with silver-stained gels.
- Improper storage of the standard or repeated freeze/thawing can result in protein degradation.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can cause smearing and this is a problem especially with silver stained gels.
- Bands will not be as well resolved in low percentage gels. Try using a higher percentage gel.
- If the bands look smeary and non-distinct after a western transfer/detection, this may be due to the antibody being too concentrated. Follow the manufacturer's recommended dilution or determine the optimal antibody concentration by dot-blotting.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- Check the gel type/percentage of the gel that was used. Depending on the gel type and/or percentage, all the bands may not be seen. For example, the smallest bands of the protein standard may not resolve on a very low percentage gel whereas the higher molecular weight bands may not resolve on a high percentage gel.
- Check the expiration date on the protein standard. Expired lots may result in faded or missing bands due to protein degradation.
- Check the storage conditions for the protein standard. Improper storage conditions will compromise the stability of the proteins in the standard.
- Make sure that the protein standard was not heated/boiled prior to loading on the gel. Our protein standards are ready to load and we do not recommend heating/boiling them as this may cause degradation of proteins in the standard.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
You can combine 5 µL MagicMark XP Standard with 5 µL Invitrogen Sharp Prestained Standard or 10 µL MagicMark XP Standard with 5 µL SeeBlue Prestained Standard in the same run using the same lane. The colored bands from the prestained standard can be used to confirm gel run and transfer. Following immunodetection, sharp MagicMark XP bands will develop directly on the western blot.
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The Invitrogen Sharp Prestained Protein Standard is suitable for NuPAGE Bis-Tris, Tris-Glycine and Tricine gels. This standard has not been tested on NuPAGE Tris-Acetate gels. The dyes that are covalently bound to the proteins in the Invitrogen Sharp Prestained Protein Standard were selected to minimize any effect they may have on migration of the ladder in different gel chemistries. As a result, the apparent molecular weights of the proteins are the same in NuPAGE Bis-Tris, Tris-Glycine and Tricine gels. Please see figure on this webpage (http://www.thermofisher.com/order/catalog/product/LC5800?ICID=search-product) showing the apparent molecular weight of each band. Note that the 3.5 kDa band is visible only on NuPAGE Bis-Tris gels run using MES SDS Running buffer or Tricine gels.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The Invitrogen Sharp Prestained Standard has not been tested on zymogram gels. We recommend using the SeeBlue Plus2 Prestained Standard (at 2-3X concentration) for those gels.
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The proteins in the Invitrogen Sharp Prestained Protein Standard are synthetic fusion proteins and their origins are proprietary. The 10, 20, 60 and 80 kDa proteins in the Invitrogen Sharp Prestained Standard have C-terminal 6xHis tags and hence are shown to cross react with the anti-His C-term antibody.
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We recommend storing the Invitrogen Sharp Prestained Protein Standard at -20 degrees C. It is stable for 12 months when properly stored.
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Yes, we recommend the Invitrogen Sharp Prestained Protein Standard (Cat. No. LC5800). It consists of 12 prestained protein bands in the range of 3.5 kDa to 260 kDa. Each band is stained in a distinct color for easy identification, and the bands are extremely tight and sharp for the most accurate molecular weight estimation. The marker is ready-to-use under denaturing and reducing conditions and can be used on NuPAGE Bis-Tris, Tris-Glycine, and Tricine gels.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is prestained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.
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Our protein standards are not designed for protein quantitation.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We do not recommend using our prestained standards for native gel electrophoresis since they are already denatured (in SDS sample buffer) and pre-reduced (by a proprietary method), and will not resolve well in under native conditions.
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We recommend using unstained protein ladders for molecular weight estimation applications as prestained ladders have a dye that is covalently bound to each protein that will result in the ladder migrating differently in different buffer systems (i.e., different gels). As a result, using a prestained ladder for molecular weight estimation will only give the apparent molecular weight of the protein. Prestained ladders may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained ladder should be used.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Please find this information in the respective manuals for the individual protein standards.
Find additional tips, troubleshooting help, and resources within our Protein Standards and Ladders Support Center.
Our protein standards are ready to load. We do not recommend heating them as this may cause protein degradation.
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Except for our NativeMark Unstained Protein Standard (designed for native electrophoresis), all of the other unstained and prestained standards we offer (Invitrogen Sharp, SeeBlue, SeeBlue Plus2, BenchMark, HiMark) have been pre-reduced (by a proprietary method). Hence, you do not need to add reducing agent.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
SeeBlue prestained standard, SeeBlue Plus 2 prestained standard and Invitrogen Sharp prestained standard are compatible with Bolt Bis-Tris Plus gels. Their migration is the same as that in NuPAGE gels with the corresponding buffer.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.