so the primary antibody we're using is particularly dirty, and even with 5+ five minute washes in TBST, we still get a good amount of background.
I know its not the blocking or secondary.
I heard that some people do washes overnight at TBST. Would this wash away any of the specifically bound antibody as well as the nonspecificbinding antibodies?
I haven't tried overnight washing in TBS-T. But I have used a high-salt wash for 30 minutes with very good results when background is dirty from the primary antibody. Here's the buffer:
0.5 M NaCl/ 0.2% SDS prepared in PBS or TBS-T.
I use it immediately after developing a film with this characteristic dirty background. Briefly rinse ECL reagent from membrane and then incubate at room temp for 30 minutes in the high- salt buffer. Briefly rinse in TBS-T to remove the high-salt wash and incubate again for 5 minutes in your ECL reagent. Expose and develop film.
I haven't tried overnight washing in TBS-T. But I have used a high-salt wash for 30 minutes with very good results when background is dirty from the primary antibody. Here's the buffer:
0.5 M NaCl/ 0.2% SDS prepared in PBS or TBS-T.
I use it immediately after developing a film with this characteristic dirty background. Briefly rinse ECL reagent from membrane and then incubate at room temp for 30 minutes in the high- salt buffer. Briefly rinse in TBS-T to remove the high-salt wash and incubate again for 5 minutes in your ECL reagent. Expose and develop film.
If you wash ON with TBS-tween you proteins will, likely, detach from the membrane, not the ab, but the ones you transfered. You say that it's not the blocking, but have u tried to increase milk or bsa? or try both? Blocking for longer periods of time? Incubating the primary for less time?
On the line of Andrei's answer, usually you can purchase the protein/peptide itself and run it next to your lanes, if you use a siRNA approach you might also KD some isoforms and then you will not know which band is the right one (I have been there).
Good luck!
No chance to get a better first antibody? We tried several recipes in similar cases and found none which is universal. Sometimes high salt is helping a little bit, like suggested by Diana, but washing o/n never improved the results.
- Badges
- Science method