During my western blot procedure, instead of sharp bands, I have been getting getting bent V-shaped, or bracket shaped bands for any low molecular weight protein (GAPDH and beta-actin, ~40 kda). This makes me less confident in quantifying my western blots and I want to determine what causes this distortion.

A previous member of the lab was able to obtain crisp GAPDH bands using the same protocol. Even the first couple of blots I did had nice looking bands. Other than getting fresher reagents, I did not change anything in the procedure until I lowered the voltage and temperature. Has anyone else experienced these changes and were able to fix them?

My transfer protocol:

I use the Biorad protein gel system. After lyses I measure the total protein and heating in SDS buffer. I add 25 ul of sample (3-5 ug total protein) to a 10 well 4-15% gel. I run at 150V at room temperature until the dye front reaches near the bottom. I activate transfer paper with methanol -> H2O -> transfer buffer. I transfer at 100 V for 1 hour with an ice pack and cold buffer and sitting on ice to stay cool. After which I block and add my antibodies.

I assume the problem is when running the gel. I've made sure to use fresh buffer and clean the apparatus. I also now lowered the voltage to run the gel to 90 V and run the gel and and transfer in the cold room. I think it may have slightly improved, but the bands are still curly shaped.

Images:

The lower bands of the first image are from the Western Blot of a previous member. The second image has GAPDH bands which were straight and not smudged which I obtained. The third and fourth images of my later GAPDH blots which came out distorted. In the last image I used beta-actin as the marker (lower bands). It too has the distorted shape.

Thank you for any help and suggestions.