low signal in WB - (Dec/03/2010 )

Hi,
I made western blot on 20ug of protein in brain tissue extract, with primary polyclonal antibody rabbit 1:500, i got strong signal with many aspecifics, then, i made another w.B on 20ug protein extract with diluted primary 1:1000, i have low signal, i can't qantify, i incubate the same filter with seconday antibody anti-Rb again for 1h, the filter looks cleaner but the signal is still low, so, i decide to make longer incubation of the same filter with primary for another overnight 4°c, im gonna develop by ECL.
im wondering about the cause of this low signal and aspecifics as well, is the primary antibody that isnt specific ?
is the seconday antibody? ...

since you didn't change the secondary antibody concentration and lost the nonspecific binding then it is most likely coming from the primary antibody.

if the response is too low then you may want to try an intermediate concentration of primary or try a longer incubation (as you are doing) or try incubating at a higher temperature (if you are incubating at room temperature then try 37C).

thanks for your interesting reply!
ive stripped 2 filters, i checked by ECL, that it was stripped well!, i incubate one filter as trial with primary at 4°C overnight, 1:500, i will use secondary anti-Rb HRP at 1:10 000, develop ECL plus and see the western!
im wondering if its worth to try later an incubation of primary at room temperature overnight instead of 4°C!
...

you can try overnight at room temperature. when i first started doing westerns we used 2 hr at 37C for the primary (1.5 hr at 37C for the secondary).