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View additional product information for NuPAGE™ Sample Reducing Agent (10X) - FAQs (NP0004, NP0009)
20 product FAQs found
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。
尽管我们推荐使用NuPAGE样品还原剂以加强稳定性，但新鲜的β-巯基乙醇可以代替NuPAGE样品还原剂并获得相同的结果。通常，终浓度为2–5%的β-巯基乙醇足以还原样品。
NuPAGE样品还原剂（10X）含有0.5M DTT（二硫苏糖醇）和独特的稳定剂。
NuPAGE样品还原剂和NuPAGE抗氧化剂应保存在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.
The formulations of buffers for our precast protein gels can be found at this link: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-electrophoresis-buffers-reagents.html
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Either BME or DTT can be used in the NuPAGE LDS Sample Buffer.
Make sure that a fresh solution of BME is used. FINAL concentration:
DTT 50-100 mM
BME 2-5%
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
TCEP, Tris Carboxy Ethyl Phosphene is an alternative sulfhydryl reducing agent for protein samples. It is an extremely potent and effective reducing agent for particularly ‘difficult' proteins. It is compatible with the Tris-Glycine gels and NuPAGE gels. It should be added to the sample buffer for these systems. 20 mM final (maximum) concentration is sufficient for samples. You may add alkylating agents, e.g. Iodine (50 mM Iodoacetic acid), to prevent re-forming of S-S bonds but it is not necessary. Do not heat because this will hydrolyze much of your sample. Instead let the sample sit for several minutes at RT and then load.
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.
Chlorobutanol is used as a preservative in the NuPAGE transfer buffer and is not necessary for efficient transfer of proteins. You may prepare the buffer without chlorobutanol but keep in mind that the buffer will not be stable for long periods. We recommend using it within 2 weeks.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We do not recommend using Carbonate or CAPS transfer buffers to transfer NuPAGE gels as the transfer efficiency will be badly compromised. Further, the high pH environment (>pH 9) of these buffers will make the NuPAGE Antioxidant non-functional.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
To increase efficiency of transfer of high molecular weight proteins from NuPAGE gels, we recommend pre-equilibrating the gel in 2x NuPAGE Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x NuPAGE transfer buffer containing methanol and 0.01% SDS.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Although we recommend using the NuPAGE Sample Reducing agent for stability reasons, fresh, neat beta-mercaptoethanol can be substituted for the NuPAGE Sample Reducing Agent, with equivalent results. A final concentration of 2-5% beta-mercaptoethanol is usually sufficient to reduce the sample.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The NuPAGE Sample Reducing agent (10X) contains 0.5M DTT (Dithiothreitol) with proprietary stabilizers .
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
NuPAGE Reducing Agent and NuPAGE Antioxidant should be stored at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.