My westerns haven't worked for the past three to four months, which is an issue. I attempted using two different antibodies from two different companies, but I got no blots. I load 50 to 100 ug of protein onto gel using SDS-PAGE concentrations ranging from 12 to 17%. I started with the Poncaeu stain until I was confident before moving on the westerns. What might be the cause of that then?
Sabine Strehl
GABARAP is a rather small protein with a MW around 15 kDa. Have you checked with a molecular weight ladder that proteins of this size are properly transferred to the membrane?
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Megha Chechi
Yes, i checked ,and in 15% gel bands are visible on half of the blotting membrane but not properly visible at the end of the blot .
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