Fuzzy bands in western blot - (Jun/23/2011 )
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
you got to give more details to give you suggestions, it will be better if you post the pic of ur blot.....i guess the prob might also be the transfer condition. how long you do transfer??, sometimes over transfer can make the low mass proteins fuzzy.....
glycoproteins will give fuzzy bands.