Immunodetection of 40-50kDa proteins

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Wed Jun 3 02:18:59 EST 1998


Hi all,
This is a supplement to the method posted below, because I was asked how to
prepare the borate buffer used in coupling.
That´s the recipe I´ve been using sucessfully for years:
(from Stoll & Blanchard (1990), Method in Enzymology, 182:24-38):
Prepare two solutions:
A: 0.2M boric acid (12.4g/l)
B: 0.05M borax (sodium tetraborate) (19.05g/l; that´s actually 0.2M in terms
of borate)

mix 50ml of solution A with 59ml of solution B to have a starting point. Make
sure the buffer is within the right pH range (pH indicator paper is
sufficient). This buffer is the 200mM borate buffer referred to in the post. 
If you want, you can titrate to exactly pH 9.0 with either of the both
solutions (add A to make acidic or add B to make alkaline). For the purpose of
coupling, no exact pH adjustment is necessary, though.

Hope this helps,
Frank



Frank O. Fackelmayer wrote:
> 
> 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,
> Frank
> 
> madavare at students.wisc.edu 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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