I am running SDS-Page western blot using 10% acrylamide gels. However, my samples are not migrating more than 55 kDa. The bands are not defined. I am using 4x Laemmli buffer with LDS from Biorad. The cell lysates are human whole brain lysates. I am wondering if the LDS has something to do with this? I tried to boil the samples at 95 degrees for 5 min; heat at 70 degrees for 10 min, all did not work.
I suggest that the problem may be that the samples contain too much lipid. You can remove the lipid by acetone washing precipitation.
https://www.researchgate.net/post/How-to-remove-excess-lipids-from-protein-samples#:~:text=You%20can%20also%20try%20to,SDS%2Dbuffer%20%2D%20SDS%20gel.
Adding on Adam B Shapiro suggestion, note that much of the sample is stuck on top of the gel. Did you add beta-mercapto/DTT to the loading buffer and did you heat the sample before running it?
You might also want to see this discussion
https://www.researchgate.net/post/How_is_possible_that_my_samples_stick_in_wells_in_SDS_PAGE
we still think it is because of the LDS content of the 4x Laemmli buffer, as all other components were checked. I am using SDS running buffer and I think 4x LDS-based Laemmle buffer might not be compatible. I will also switch to DTT instead of beta-mercapto to rule out otherwise. I will keep you updated.
Did you manage to resolve the problem? I'm going through the same.
The problem was with the Human Brain Whole Tissue Lysate (Adult Whole Normal), novus. When compared with colorectal cell lysate, this difference was obvious. Thank you all for your comments.