antibody titer - (Dec/24/2008 )
hello all, still having problems with the ChIP
a co-worker stressed it is very important to empirically determine the proper antibody concentration to use in order to get the best results
so how would one go about this? i have so far just been adding 5 micrograms and incubating overnite, my logic was if this was a small amount the overnite incubation should cover me, but i am not sure what the negative effects will be if i am adding way too much (maybe i only need 1 microgram?) what would too much antibody do to my results?
i do not have a very large supply of my starting material (primary keratinocyte culture), so must be very conservative in my approach unfortunately, but if i did have an endless supply how would one perform and verify an antibody titer?
enjoy the holidays
a co-worker stressed it is very important to empirically determine the proper antibody concentration to use in order to get the best results
so how would one go about this? i have so far just been adding 5 micrograms and incubating overnite, my logic was if this was a small amount the overnite incubation should cover me, but i am not sure what the negative effects will be if i am adding way too much (maybe i only need 1 microgram?) what would too much antibody do to my results?
i do not have a very large supply of my starting material (primary keratinocyte culture), so must be very conservative in my approach unfortunately, but if i did have an endless supply how would one perform and verify an antibody titer?
enjoy the holidays
If you want to titer the antibody but don't want to use up too much of your chromatin you could always try decreasing the volumes of chromatin, dilution buffer, beads, elution buffer, etc. You may not be able to run very many PCRs with the resulting material but you don't really need to if you are just looking at one or two genomic regions.