Hi!
I met some issues with my western blots recently and I don't understand why. The target protein is AMPKα1. As you can see in the pic, there have two bands, but this product reference shows there has only one band.
Any kind of advice would be much appreciated. Thank you!
It could be caused by a proteolytic cleavage producing the smaller band from the larger one; read-through of a stop codon producing a protein that is longer than expected; alternative splicing resulting in two protein products from the same gene; or non-specific binding of the antibody to a second protein in the sample.
Adam B Shapiro Thank you for ur answer! Maybe the time I used RIPA was too long, around 50 mins. Another question is whether I can use the down band in my figure.
Dear Zhizai Chen
AMPKα1 is a AMP-activated protein kinase (AMPK). When it is catalytically active, it is phosphorylated by upstream kinases (AMPK). In western blot, if a protein is hyper-phosphorylated, it is usual that to obtain more than one bands. The higher molecular weight band in your blot was coming from phosphorylated state of AMPKα1. Prepare a control sample treated with phosphatase without providing any phosphatase inhibitor in the lysis protocol, you may overcome the double band.
I am attaching one blot image of AMPKα1 here. Check the link.
https://www.bosterbio.com/anti-ampk-alpha-1-antibody-a00994-3-boster.html
Best wishes.
Dear Satyendra Mondal thank u for your answer. These proteins I treated with RIPA+1%cocktail to lyse. And the pic I showed those bands are between 70-55 kDa, especially the up band is near 70 kDa, so I don't think it is p-AMPKa1.
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