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I am very frustrated at myself because I can never get a western blot right....and it's a diff problem everytime
So this time I am blotting for a protein with different treatments in diff cell lines. I do get bands around my expected size but there very fuzzy and I can't tell which one is the correct one. My protein is about 39kd but with my antibody I get a band a little lower than 50kd with a 8% gel. I used to get a sharp clear band but recently I have been getting multiple fuzzy bands. I've been using the same vial of antibody and I thought multiple freeze-thaw might be a problem. However, when I load samples from different experiments I get get crisp bands in some samples and fuzzy multiple bands in others. I processed all the samples the same way (e.g same lysis buffer, loading dye, heating etc.). I read somewhere that glycosylation of protein can give fuzzy bands and my protein of interest does get glycosylated. However I don't have much knowledge about that so I am asking for your expert opinions and suggestions. Please help me!!!!!
-disillusioned first year grad student
