Immunodetection of 40-50kDa proteins

Frank O. Fackelmayer fof1 at
Tue Jun 2 02:18:01 EST 1998

Hi Monika,
I had best results by covalently coupling the antibodies to protein-A
sepharose before IP, using DMP (dimethyl-pimelimidate). They can´t elute from
the column then.  
Plain simple, and works very reproducibly. That´s how:

1. prepare affinity-purified antibodies (by an antigen column), or
IgG-fraction (by chromatography over protein A sepharose).
2. incubate your antibody with protein A sepharose (2h to overnight at RT) in
any buffer that´s suitable for western blots (e.g. TNT, TBST...). The ratio of
antibody to volume sepharose can be varied. A good starting point is 1ug per
10ul of sepharose. I usually prepare between 200ul and 1ml of antibody-column
in one batch, that´s handy.
3. wash well with binding buffer.
4. wash well with borate buffer (200mM, pH 9.0) to remove all amine-containing
buffer substances, e.g. Tris, that would inhibit coupling. Use at least
50volumes of borate buffer for washing. Washing is best done in a disposable
plastic column, e.g. a poly-prep column (biorad). Perform next steps also in column.
5. resuspend sepharose in 10 volumes of borate buffer.
6. add solid DMP (Sigma) to a final concentration of 20mM, mix immediately,
let stand for 30min at RT.
7. drain well.
8. repeat step 5 to 7, but incubate for only 5min.
9. wash well again in borate buffer.
10. add 2 volumes of 1M Tris to quench coupling reaction, incubate for 2h with
gentle mixing.
11. wash in buffer for IP (or TNT, TBST...)
12. mock-elute sepharose with 200mM NaCl, 200mM Glycine, pH 2.0 to remove all
non-covalently bound IgGs.
13. repeat step 11 until neutral.
14. store at 4C with traces of merthiolate. Stable for several months
15. Use for IP.

Hope this helps,

madavare at wrote:
> I am interested in a putative association of  45-55kDa protein with my
> favorite protein.  I'm trying to show this in vivo (tissue extracts) by co-
> immunoprecipitating it with my protein (antibodies to IP is pAB and probing is
> mAB). I am detecting these by western blotting (secondary reagent is goat
> anti-mouse (IgG)- HRP).  Ofcourse, the huge IP antibody (due to secondary ab.)
> signal precludes any conclusive data for my protein.
> Are there methods to block the IP antibody prior to probing.
> I would appreciate any comments/suggestions/advice about this, especially if
> anyone has resolved this issue successfully.
> thanks!
> Monika
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