Dear all,
I have a protein with two isoforms, separated by 5-6 kDa. The size of these isoforms is about 80 kDa for one of them, and around 85 KDa for the other.
I was wondering whichso percentage of the SDS-PAGE I can use in other to obtain the maximum separation for booth isoforms.
Also, I was wondering about another advice, like running the gel at a slowly and constant voltage.
Thank you sou much!
Best,
Miguel
Tris-tricine buffer system is recommended by using 18%T and 5%C.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa by Schagger and Vonjagow 1987.
I would do it in 12% or higher with SDS buffer. Also, longer run with higher SDS helps better separation of bands.
Dear Miguel,
To achieve maximum separation of your protein isoforms, you can consider using a higher percentage SDS-PAGE gel. Higher percentage gels are more suitable for resolving smaller-sized proteins. In your case, with isoforms separated by 5-6 kDa, using a higher percentage gel will help in better resolution.
Typically, a 12% SDS-PAGE gel is a common starting point for separating proteins in the range of 10-100 kDa. However, since your isoforms are around 80-85 kDa, you might want to try using a slightly higher percentage gel, such as a 15% or even a 17.5% gel. These higher percentage gels will provide better resolution and separation for your isoforms.
Regarding the running conditions, a slowly and constant voltage can help with better resolution and band sharpness. Running the gel at a lower constant voltage can minimize heat generation and prevent excessive band smearing or distortion. You can consider running the gel at around 100 V or lower to achieve a slower and more controlled migration of the proteins.
Additionally, you may also want to optimize the running buffer composition, such as using Tris-glycine or MES buffer, to further enhance the separation of your protein isoforms. Buffer pH and ionic strength can also influence the migration behavior of proteins.
It's important to note that these suggestions are general guidelines, and optimization may be required based on the specific characteristics of your protein and the gel system you are using. It's recommended to perform a few test runs with different gel percentages and running conditions to determine the optimal conditions for maximum separation of your protein isoforms.
Best of luck with your experiments!
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
Dear Miguel,
in principle, I agree with the previous speakers, but I would propose a different approach to the problem in order to achieve the best possible result. Tricine-PAGE gives best separation for smaller size proteins, due to its difference in pore size compared to SDS PAGE. You can find more information here:
Article Tricine-sds-page
Chapter Tricine-SDS-PAGE: Methods and Protocols
https://www.nature.com/articles/nprot.2006.4
If you can't buy precast gels (the best choice) here you can find a receipt:
http://www.oardc.ohio-state.edu/stockingerlab/Protocols/Tricine%20Gels.pdf
Best regards
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