how much purify protein nedded for IP - (Apr/24/2014 )

hi

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.

 

thanks

 

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.

hi

 

thanks

 

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.

 

thanks

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.

 

http://www.abcam.com/?pageconfig=resource&rid=11385

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.

thanks  guys

 

if I do that how much of Protein should I use. 2ug 5ug 10ug or more?

 

thanks

that depends on how much antibody-protein a agarose you have. you may have to determine the capacity empirically.