I am running a NuPAGE 10% Bis-Tris mini gel using mouse white adipose tissue (WAT) whole cell extract as my sample, and after staining my membrane with Ponceau Red, I'm seeing thin, vertical bands in most of my wells except for the last 2 or 3 (image attached). I've been told that working with WAT can sometimes be a pain, but I was wondering if anyone has any ideas what could be causing the irregular vertical banding pattern? I loaded 40 ug in each well.
Thank you so much!
Hi Lauren,
Usually vertical streaking would be due to high protein loads. Have you tried loading lesser amounts of protein.
This also could be due to drag caused by lipids. Try adding more of detergents along with with SDS (May be 0.1 -0.3% of Triton-X100) to prevent interference due to lipids.
Hi Lauren,
Usually vertical streaking would be due to high protein loads. Have you tried loading lesser amounts of protein.
This also could be due to drag caused by lipids. Try adding more of detergents along with with SDS (May be 0.1 -0.3% of Triton-X100) to prevent interference due to lipids.
I agree with Rakesh, it is mostly because of the lipids. When you lyse cells and the lysate is filled with lipids and DNA, it forms a gunk which runs very similar to what you see. My opinion, try to spin down the heavy lipids or increase the concentration of your detergents.
Agreed. Excess lipids can cause vertical streaking. Would recommend spinning more and also, freeze thaw few times before you spin.
You can also try to precipitate your proteins in cold acetone to get rid of lipids. You should get a protein pellet (after centrifugation) that can be resuspended in sample buffer and run electrophoresis immediately.
Hi Lauren, at what voltage you are running the gels? It seems the proteins might have migrated too fast.
Hello Lauren,
I encountered the same problem and in my case, it was the precooled gel wells that were dirty. I think that waste (gel residues) in wells can negatively impact migration. By cleaning the wells by suction-discharge by pipette with migration buffer before use, it is possible to flush the small pieces of gels and solve the problem.
Good luck!
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