western blotting problem with detection - signal fades away quickly (Sep/14/2009 )
Hi all,
I have done a western blot recently. I am using supersignal pico from Pierce as a substrate. for the initial few exposures, there is a lot of signal and consequently large background. Especially my biotinylated marker and beta actin look like a smear. this also showed my probable bands of interest. however, on further exposure those bands disappeared. the delay from the first exposure was less than half an hour. apparently, the pico is supposed to last for good 8 hours. a prolonged exposure gave me nothing except few "spots" on the film. this is my second encounter with pico, before that i used dura and it sort of gave similar results. I am wondering is there a problem with my secondary antibody ? (frozen aliquotes). does the HRP degrade rapidly? maybe cos of thawing? or some other reason? I am getting frustrated with western blots now mainly cos they are not working and at it since few months now!!
any help would be highly appreciated.
Thanks in advance!
maybe your secondary antibody is too concentrated and you're using up the substrate too rapidly.
cupidstunt on Sep 14 2009, 08:37 AM said:
I have done a western blot recently. I am using supersignal pico from Pierce as a substrate. for the initial few exposures, there is a lot of signal and consequently large background. Especially my biotinylated marker and beta actin look like a smear. this also showed my probable bands of interest. however, on further exposure those bands disappeared. the delay from the first exposure was less than half an hour. apparently, the pico is supposed to last for good 8 hours. a prolonged exposure gave me nothing except few "spots" on the film. this is my second encounter with pico, before that i used dura and it sort of gave similar results. I am wondering is there a problem with my secondary antibody ? (frozen aliquotes). does the HRP degrade rapidly? maybe cos of thawing? or some other reason? I am getting frustrated with western blots now mainly cos they are not working and at it since few months now!!
any help would be highly appreciated.
Thanks in advance!
I agree, it sounds as if your secondary is too concentrated. How much are you using? Pierce recommends using your secondary at 1:20,000 to 1:100,000 and possibly using less primary as well (start at 1:1000). They also recommend six washes instead of four and incubating the blot with the ECL mixture for 5 minutes instead of the 1 minute used for normal ECL.