Monoclonal antibody vs. polyclonal antibody

[snowysummer]

I have a question,

 

How come if we immunoprecipitate with monoclonal antibody, then we should blot with polyclonal antibody, or the other way around?

 

Thx ~

[zyncod]

Well, if you IP with a monoclonal, then you've already bound up your mAb's binding site. You can IP and blot with a poly, though.

[scicop]

I have a question' date='

 

How come if we immunoprecipitate with monoclonal antibody, then we should blot with polyclonal antibody, or the other way around?

 

Thx ~[/quote']

Then, its not necessary to use different mono or poly, providing that you're using antibodies from different species.

 

It was "suggested" that you IP with a mono, since it is very antigen specific (i.e. to one small peptide in the protein) and use a poly to blot since a poly (is really multiple antibodies that can recognize multiple antigens (peptides) on the same protein) can increase detection sensitivity with older chemoluminescent techniques. (this is your answer)

 

However, this is really no longer the case, there are really good CL kits out there that allow you to detect antibodies with low avidity and low detection seen with monoclonals, despite specificity.

 

Also,alot of monoclonals today are being produced in ascites, which product some really concentrated stock solutions of mAB! So, its not really a big deal anymore to IP with one or the other...so long as its different species, you're OK.

[snowysummer]

thx a lot~