Help needed with western blot with human serum - (Feb/07/2007 )
i`ve got a problem with wb using human serum. i use ripa for cell lysis and then run nupage gel. block membrane with 4%bsa and incubate with different dilutions of human serum to get best conditions. the point is when i use normal serum against normal eg neutrophils, and secondary IgG anti human, i have got a lot of bands!!!! i expect some of them but not so many. i used to develope membranes on kodak films but i have switched to alpha inotech imaging system. i do remember not seeing any additional bands when using films (except l+h chains if igg), and now quite few. i found this system more sensitive, but i don`t think this is the cause of many bands. did anybody try wb with human sera? i tried different secondary but same result..... i attache the pic of normal serum (dilutions) against KG1 cell line lysate - i found it same when used neutrophils.
What's about dilution of secondary antibodies (to avoid nonspecifity I use 1:10000 or even more.
thanks lot... i`ll try to do it.