IP problems - The antibody doesn't bind to the beads (Dec/21/2007 )

Hi,
The aim of my experiment is to pull down my protein of interest (the endogenous one from a lysate) to find its physical interactors.
I’m trying to bind covalently the specific antibody to protein A beads: I’m using DMP as the cross linking reagent (20 mM in 0.2 M sodium borate pH 9.0 buffer) and 0.2 M Ethanolamine-HCl, pH 8 as the quencing solution. The antibody doesn’t bind covalently to the beads: I find it in the elution sample if, after the binding reaction, I wash the beads with Glycine 100mM pH 3.0.
I thought it didn’t work because my antibody solution contains amines (Tris and glycine), so I dialysed it against a PBS buffer. I did the experiment another time, but I had some problems with the borate buffer because it precipitated at room temperature (20°C) and the antibody didn’t bind to the beads.
Could anybody help me? Any suggestions? (My antiboby is a Guinea pig one and I incubated it with the beads o.n. at 4°C)
Thanks in advance for your kind help

Drosy

did you try the beads after the binding? finding antibody in the wash is normal. you will not get 100% binding and you also should have a large excess of antibody to protein a molecules.

if you aren't seeing any binding then you can try protein g. if that doesn't work then you can try ah- or ch-agarose (the antibodies won't orient as well but should bind).

Suggestions:

1. Bind Ab to protein A/G beads at room temp (in whatever buffer it's in), then do the following at 4C:
Wash 3x w/ PBS to get rid of free amines (Ab will stick to protein A/G)
couple in a small volume of PBS w/o amines for an hour or so (while rotating)
quench unreacted functional groups with an equal volume .5MTris or Glycine (beaucomp free amine groups)
wash a few times

THEN, Check to see if the Abs bound by:
Boiling in SDS-Page sample buffer:
a uL or so of Ab + reducing agent
a few ug of beads post-coupling in NON-REDUCING CONDITIONS (who loves to make new sample buffers? you do!)
a few ug of beads post coupling + reducing agent

Scale the volumes you boil to equivalency, so that you're loading the same fraction of the total reaction in each lane. (EG: 1/50th of total Ab used, 1/50th of beads).

Run them out on a SDS-PAGE gel and silver stain.
If your coupling reaction worked, you should see:
Ab: 25kDa + 50kDa IgG L + H chains (plus some serum smear around 50-55kd)
non-reducing beads: Protein A, Protein A+Ab fragments
Reducing conditions: same as above, but look for liberated light chains at 25kDa.

If you don't see lots of light chains in the last lane, your coupling failed.
Which means...
1. Go buy new coupling agent, yours may have lost activity. DSS/EDC/Etc.
2. Try coupling at a lower pH - 7-8 should be fine. pH 9 seems a little ... off, biologically speaking.

Other notes:

If you're trying to determine interacting partners, use crosslinked ("CL") sepharose beads instead of agarose beads. The sepharose beads will survive boiling, allowing you to remove your protein mix spnt from the beads/protein A. Using protein-A-agarose (something I did about two weeks ago) is fine, until you boil the beads, they dissolve, and you get a fat protein A band on your final gel. They're a bit pricier, but ultimately worth it, as you don't run into problems with protein A/G masking your interacting partner.

Let me know if anything works;
~TheRak

Dear Mdfenko,
I beg pardon for my late reply, but I was ill.

I didn’t try the beads after the binding, but I boiled them in SDS-Page sample buffer and checked for the presence of the antibody by W.B.
I loaded 1/50 of the beads (taken after the coupling, but before the elution step) and I didn’t see any signals on the WB filter ... Perhaps I loaded few beads (2.5 ul of the 100 ul total beads).
To test, by WB, if the beads work I have a problem: the molecular weight of the protein I’m trying to pull down is 50 KDa.
I think I should prepare a lysate with a tagged form of my protein, or I should buy a HRP-conjugated Protein A (like a secondary antibody it binds the Fc portion of the primary antibody but, unlike the secondary antibody, doen’t bind the denatured individual HC and LC polypeptides on WB filters) but I don’t know anybody who has used them.

Thank you very much for your intervention!
Drosy

Dear Therak,
I beg pardon for my late reply, but I was ill.

I’ll try your protocol, but I have a practical question: during the quenching step .5MTris or Glycine means 0.5M?
I think I should buy a new coupling agent: even if nobody had used it before I did (I opened the bin), the DMP has been stored at -20°C for 3-4 years.

Thanks a lot for your intervention!
Drosy