Hello! I am getting some smearing appear on these 4-20% gradient gels. I loaded 20ug of protein each time. I am seeing my bands fine but the rest of the gel is a bit of a mess. I have attached an example blot of what I am seeing. In this blot I do believe that I loaded too much protein however there is another person in my lab using the same gels and has been having the same issue even with loading 10ug of protein. Any suggestions? Thanks!
Hi,
Perhaps due to unevenly pouring the gel? There is uneven polymerization throughout the gel causing smearing. Since you and your colleague are loading different sample amounts and getting the same results, I do not believe overloading is the issue.
Here is a link outlining possible solutions:
https://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/smear.html#:~:text=Smeared%20gels%20%E2%80%93%20example%201%20Smearing%20can%20have,%20or%20due%20to%20gross%20overloading%20of%20protein.
Hope this helps!
Kimberly Edicha
Ashten Stambersky gave a good link. Without understanding polyacrylamide gel electrophoresis and SDS, it is difficult to make a good experience.
1.Polyacrylamide slows down protein diffusion.
2. SDS binds each amino acid at an amine group.
3. Therefore, the pH should be less than 7.
4. For each amino acid of the polypeptide there should be one SDS molecule with a slight excess
5. Uniform distribution of SDS in the polyacrylamide gel is necessary.
These are actually precast gels, so I personally did not pour them myself.
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