I am doing western blots for UCP1 in mouse BAT and I have not had problems with previous blots, however, recently all my blots have had the same streaking in the bands. Below are pictures of one of my previous blots and one that I did today. I am not sure as to why this is occurring. I use 12.5ug of protein in each well, and use 5%skim milk blocking. I haven't changed my protocol in anyway either.
Each lane is whole tissue lysate from a different mouse. I was thinking it could be a problem with the length Of the stacking gel, But I was also wondering if it could be a problem with the polymerization of my gels?
Also, when I transfer my protein, I cut the membranes after so I can blot for alpha tubulin additionally. The UCP1 protein in the pictures is about 32kDa. I don’t see any problem in the alpha tubulin blots when I do this. I did this with all 4 blots in the pictures.
Hi Erik,
was it a fresh preparation? I have also suffered this problem with old samples. Change the PI and follow the other recommendations.
Fast electrophoresis causes streaking.
Did you boil your samples in sample buffer? Although that is the standard procedure you may get better results if you heat to 60 °C only, for 10-15 min.
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