Homemade DNA/Protein A beads - i think i made a boo-boo (Jun/22/2005 )
So I did something stupid, not paying attention, and made my own protein A/DNA beads for ChIP in 10%BSA/NaN3/TE rather than 0.1%BSA/NaN3/TE. Unfortunately, I didn't notice my error until after I had already pre-cleared my sonicated supernatant with them.. Whats everyone's opinion about 1) whether these beads would work at all for the IP step (are they "overblocked" or something)? and 2) would addition of all that extra BSA into the tubes mess up the expt. even if i make new beads for the pulldown step?
thanks in advance for sharing!
Well, I think most likely it will not ruin the experiment.
I mean it could be argued that overloading wih BSA might increase nonspecific
binding to antibody thus masking it from binding its epitope ......etc.
But, as long as you have good positive controls for your ChiP (and a negative) I wouldn't worry too much about it. If you have proper controls it will be obviously (knock on wood) if your experiment worked well with the extra BSA.
thanks for the reply. for those who are keeping score, the expt works (+ controls are positive, negative controls are negative) , even with all that extra BSA (although I did make new beads for the pulldown).