question about antibodies & protein G/A sepharose in CO-IP - (Mar/12/2006 )
Hi,
I am about to do a CO-IP in this week, however, I just found that I ordered Protein A sepharose for mistake.
My CO-IP is for HA-GSK-3beta and Tax, I planned to use anti-HA antibody to precipitate HA-GSK-3beta and then use anti-Tax antibody to detect the presence of Tax. Our HA primary antibody is rabbit polyclonoal and the Tax primary ab is mouse monoclonal. As I said, I have already ordered protein A sepharose. I'm kind of anxious to do a CO-IP, so I'm thinking about using anti-Tax antibody to precipitate Tax first and then detect the presence of HA-GSK-3beta by immunoblot? Do you think the second alternative method could work as well as the first one?
thanks,
sorry about my bad English
Hi Ivytony-
Uh...do a quick google search for something like antibody affinity for protein A/G. You should quickly find a graph of different species and different antibody isotype's affinity for protein A vs. G. I have a copy of said table taped to ourr lab fridge as it's a useful reference.
Generally protein A & G both bind most antibodies fairly well. The only problem I can forsee would be if your mouse monoclonal happens to be the IgG1 isotype which binds poorly to protein A (and much better to G). Even still however, you can still do IP's with protein A sepharose, you just need to use a bit more.
Good Luck,
Mountainman
Uh...do a quick google search for something like antibody affinity for protein A/G. You should quickly find a graph of different species and different antibody isotype's affinity for protein A vs. G. I have a copy of said table taped to ourr lab fridge as it's a useful reference.
Generally protein A & G both bind most antibodies fairly well. The only problem I can forsee would be if your mouse monoclonal happens to be the IgG1 isotype which binds poorly to protein A (and much better to G). Even still however, you can still do IP's with protein A sepharose, you just need to use a bit more.
Good Luck,
Mountainman
Thanks Mountainman
I just found this info about protein A agarose (sorry about my typo of sepharose in my original post) on the seller's website:
Comments: Designed for immunoprecipitation applications. This product is blocked with BSA to reduce non-specific binding and cannot be used for purification; best for mouse IgG2a and IgG2b and rabbit IgG. As it mentions protein A agarose can be used for rabbit IgG, does this mean I can use Protein A for rabbit polyclonal antibody??
thanks,
IVYtony
IVYTONY,
Yes... you can use Protein A agarose for rabbit IgG !
Wiz
In your polyclonal, you might have IgG. It should work.
The strength of interaction between the mAb and Protein G or Protein A is an important factor in the decision of which slurry to use. Protein G coupled to some insoluble matrix (e.g. sepharose beads) binds well to most subclasses of rat immunoglobulins and mouse IgG1, while Protein A binds much better to mouse IgG2a, IgG2b, and IgG3.
Binding Characteristics of Some Immunoglobulins
Immunoglobulin Protein A /Protein G
Mouse IgG1 + /++
IgG2a +++ /+++
IgG2b ++ /++
IgG3 + /+++
IgM - /-
IgA - /-
IgE - /-
Rat IgG1 + /+
IgG2a - /+++
IgG2b - /++
IgG2c + /++
Human IgG1 +++ /+++
IgG2 +++ /+++
IgG3 - /+++
IgG4 +++ /+++
in my opinion, your second alternative method would work more well
good luck