I've done several experiments using western blotting to detect extracellular vesicle markers TSG101 and CD9. However, I consistently observe bands that are larger than the expected size. Notably, my lysate shows the correct band size, but the samples consistently display larger band sizes despite the use of a reducing agent. I've used 1% SDS lysis buffer along with protease inhibitor for sample preparation, and I also attempted using RIPA buffer. Interestingly, the bands only appeared when SDS was used, but they differed in size compared to the expected bands. Any help would be greatly appreciated!
Thanks!
add the B-Me in the loading buffer and denature at 95C for 10 minutes spin down and load right away. I would process the samples in a similar fashion and perhaps the # samples need to be lysed longer. also your ladder indicates that your gel is maybe setting too fast or is jot homogenous. so I would be more careful in pouring the gel that it is evenly mixed before putting in the glass
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