Hi! I have trouble with the SDS-PAGE. I do not know why my protein band and size marker are not visible in the high molecular weight above 50 kDa.
At this moment, I have tried to change the SDS-Running buffer, but as you can see in Figure I attached the result is the same. Furthermore, i am following the online protocol of Sigma (https://www.sigmaaldrich.com/VN/en/technical-documents/protocol/protein-biology/gel-electrophoresis/sds-page).
Could you show me the way to improve it?
You may just need to use a higher percentage of acrylamide. What percentage are you using now?
Multiple reasons could exist why your protein band and size marker are not visible in the high molecular weight region during SDS-PAGE. Here are some suggestions to improve your experiment:
I agree with @Safir Ullah Khan.
The Acrylamide gel may not have run or stained proplerly. The protein sample could be diluted or denatured.The pH could be off. Proteins that are to be analyzed by SDS-PAGE should be denatured in a buffer with a pH of 8.0-9.0.
Hi there Adam B Shapiro !!
I use 10% for the resolving and 8% for the stacking gel.
Last week, everything was normal (I mean the SDS-PAGE result was ok with full bands). But I do not know why this week the result is like that.
The choice of gel percentages will vary depending on the size of the proteins you are trying to separate. For example, if you are trying to separate small proteins, you may want to use a 4% resolving gel and a 2% stacking gel. Conversely, if you are trying to separate large proteins, you may want to use a 15% resolving gel and a 10% stacking gel.
John Odonnel Yes, thank you. But as I mentioned before, last week the result was good, and this week it is not as good as expected.
I think the problem comes from the reagent eg., sample buffer, casting buffer, SDS, APS,...
Do you think the same?
Please reassess your procedures.
Insufficient sample preparation, incorrect gel concentration, improper voltage and running conditions, and contamination can cause erroraneous results.
If you are still having trouble, you may need to consult with a protein electrophoresis expert
It is possible that the protein is not properly dissolved in the sample buffer or is not running on the gel correctly. Additionally, if the protein is highly charged or insoluble, it may not move through the gel properly or be visible on the gel. It is also possible that the protein is being degraded by proteases or denatured by heat. If possible, it is recommended to run a sample with a known concentration of the same protein to confirm that the gel and sample buffer are working correctly.
Hi, i can't detect the protein marker in your PAGE. If you have used a protein marker and it does not give a band, it is probably either damaged or expired. If you have not used a protein marker, it is not possible to confirm exactly what the percentage of your gel is.
I hope it may help
Good luck
Use 12% gel and increase the protein to 10ul. I think this helps
Hi guys, I have solved this problem by changing the resolving (separating) buffer and using 12% acrylamide gel.
Thank you for all of your advisements.
There are several factors that could contribute to the lack of visibility of higher molecular weight proteins in SDS-PAGE. Here are some possible causes and solutions:
Optimizing SDS-PAGE may require some trial and error to determine the best conditions for your specific protein of interest.
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
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