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查看更多产品信息 NuPAGE™ MOPS SDS Buffer Kit (for Bis-Tris Gels) - FAQs (NP0050)
18 个常见问题解答
NuPAGE缓冲液应保存在4–25℃。
•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
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。
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.
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.
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.
One can use 0.002% thioglycolic acid in the upper buffer reservoir. This is a good scavenger of free radicals. The reference to this is described by Hunkapiller et al, Methods of Enzymology, (91), 399, 1983. Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to become oxidized itself over time. Oxidized TGA will actually promote sample re-oxidation.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Copper or zinc staining is a rapid, sensitive method for detection of protein bands . ~10 ng reduced BSA on NuPAGE Bis-Tris gels can be detected with both the copper and zinc stain.
Copper Stain: Staining solution - 0.3 M CuCl2
After electrophoresis, remove the gel from the cassette and equilibrate the gel in 100 mL of 1X running buffer* for 15 minutes. Immerse the gel in 100 ml of 0.3 M CuCl2 solution for about 5 minutes (the protein band will appear as a negative stain with a blue background).
Zinc stain: Staining solution - 0.2 M Imidazole and 10 mM ZnCl2
After electrophoresis, remove the gel from the cassette and equilibrate the gel in 100 mL of 1X running buffer* for 15 minutes. Place the gel in 100 ml of 0.2M Imidazole solution for 10 minutes. Next immerse the gel in 100 ml of 10 mM ZnCl2 solution for about 5 minutes (the protein will appear as a transparent band with a white background).
*The 1X running buffer can be the buffer from the electrophoresis tank after run (MES, MOPS). However, for better contrast of the band, the 1X Tris-Glycine SDS running buffer is recommended.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We do not recommend using gels past their expiration date because over time, the polyacrylamide hydrolyzes to acrylic acid and ammonia and this will affect the resolution of the proteins. Breakdown of polyacrylamide matrix is identified by:
- Ghost bands and doublets, seen first in the high molecular weight proteins
- Smiling of dye front across the gel, with bands in outer lanes becoming very slanted - proteins run slower there due to change in pH and pore size over time.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
This is not really recommended, but it is always possible to increase run time by lowering the voltage of the run. In general, the relationships are linear - i.e., decreasing voltage by half will double the run time.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
If you are running the gel at constant voltage, you do not need to increase the voltage regardless of the thickness of the gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Using constant voltage allows the current and power to decrease during the run, providing a safety margin in case of a break in the system. Having lower power is a safety benefit and will also decrease the chances of experiencing an overheating of the gel. Further, the constant voltage setting does not need adjustment to account for differences in number or thickness of gels being run.
Find additional tips, troubleshooting help, and resources within ourProtein Gel 1D Electrophoresis Support Center.
NuPAGE buffers can be stored at 4-25 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.
*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.
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.