High background in Western blot - (Apr/23/2004 )
Regarding the dilutions of the secondary they can go as high as 100,000. The type of membrane used does not have any significant effect according to me for both nitrocellulose and PVDF work equally good for me. try using high salt buffer for your washes (containing 0.5M -1M NaCl). This helps is reducing unspecific binding. you could alos try using coupling solution for diluting your antibody. By coupling solution i mean diluting it with 1% sera.
Hopefully this helps you.
i'm using a membrane based colorimetric detection method [sigma's proteoqwest system] and i get unbelievably high back ground, so high that i have to stop the reaction after 3 minutes of the whole membrane would be purple.
i block with their blocking col (3% BSA) overnight at 4 degrees and always use tween in my wash buffer.... any ideas? would marvel (non-milk fat) be better?
I had the same problem with anti-goat HRP secondary antibody. I tried to optimize it by using different diluted anti-goat (from 1:100000 to 1:10000) and using 5% milk or 3% BSA without primary antibody. What I found is that 3% BSA yields much much cleaner background when I use 1:100000 diluted anti-goat. In addition, I always block the blot for 2 hrs only @RT which means that you have to get up early to run gel, transfer, block and blot:-(. but good thing is that you can get the result in one day.
weird eh, i'm now using 5% non fat milk and am getting no background at all, even using my secondary at 1:1000 dilution!!!!! finally got it to work after 2 months of optimisation!
The washings between each step should be appropriate. The background may even evolve from the Luminol in the aged ECL kit.
Now I am waiting for the rabbit sera from Jacksonimmuno lab. Hopefully get some results on this weekend.
Did you try the rabbit serum as blocking solution?? I have the same problem with a rabbit anti-goat from dako, and I don't know what can I do!!!!