how much purify protein nedded for IP - (Apr/24/2014 )
I want to do an Ip of my protein. ussually when I do from a lysate I use 500ug of my total protein.
In this case I have the pure purifiy protein. So how much of protein houd I use? is 10ug good.
aslo for the IP usually people mixe the Primary antibody with the protein for 2 H or O/N, then add the protein Aagarose bead for 2 hours.
is it possible to fo first antibody+ bead O/N then next day the protein.
Consult the data sheets for your antibody and/or purified protein (assuming you purchased them). They usually indicate the amount to use for these type of assays.
not a commercial one and no info on the antibody for IP
waht about my second question
antibody binding to protein A is rapid. overnight incubation is excessive.
ok but i am not talking about the time but the sequence order.
Usually people mixe the Primary antibody with the protein for 2 H or O/N, then add the protein A agarose beads for 2 hours.
What about first antibody+ bead 2H then add the protein.
Sure, it can be done that way. I would consider doing this if co-elution of my antibodies (heavy/light chain) had a size similar to my target protein. This method has a draw back in that it will yield less protein; however, since you are using a purified protein product invitro it may not matter that much.
Edit - Found Abcam's Immunoprecipitation protocol, 4.2 method A and B.
yes, you can do that.
there are kits for preparing immunoaffinity columns. with some of them (the ones i like) you bind (then crosslink, but you're not asking about that) antibody to immobilized protein a or g. then you use this to capture the protein.
since protein a will bind the non-reactive tail of the antibody (Fc), it doesn't interfere with antibody binding.
the only thing that i would be concerned about is ensuring that the beads remain in suspension during binding.
if I do that how much of Protein should I use. 2ug 5ug 10ug or more?
that depends on how much antibody-protein a agarose you have. you may have to determine the capacity empirically.