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Antibody-beads crosslink - troubles (Jun/15/2009 )

Dear all,
I'm using the following protocol for crosslinking an affinity purified antibody to protein A sepharose beads but my antibody doesn't seem to work after crosslink (while it does if I do not crosslink). Can any of you help me? How can I solve the problem?
Thanks in advance!



Preparing Protein A Sepharose

Gently resuspension of the medium
Remove 10 ml of 50% slurry each from the container and transfer to two 15 ml Falcon (5 ml each)
Centrifuge 3000rpm/2 min
Decant and dispose supernatant
Wash twice with 10CV Na2B4O7 buffer
Centrifuge 3000rpm/2 min
Carefully decant the supernatant and discard
Resuspend the medium (agarose) in one volume of Na2B4O7 buffer
Store the equilibrated Sepharose at 4C



Cross-linking
!all steps at RT!
Add antibody (10g for each IP)

End-over-end rotation for 1h/RT
Centrifuge 3000rpm/2 min
Wash 3 times with 10CV of Na2B4O7 buffer (500l)
Centrifuge 3000 rpm/2 min
Remove 10l beads as control before cross-link
Resuspend in 10CV of DMP solution (500l)

End-over-end rotation 45 min at RT
Centrifuge 3000 rpm/2 min
Wash twice with 10CV ethanolamine (500l)
Centrifuge 3000 rpm/2 min
Resuspend in 10CV ethanolamine and rotate 2 hours at RT (500l)
Centrifuge 3000 rpm/2 min
Wash 3 times with (PBS 500l)
Centrifuge 3000 rpm/2 min
Resuspend in PBS (50% slurry50l)

Remove 10l beads to check cross-link


Cross-linked beads can be stored ON for IP. It is preferable to prepare them fresh for each experiment.



After the immunopurification antigens can be eluted with
1CV of 100 mM glycine, pH 2.5 (add, spin and collect), then add 2CV of 500mM glycine pH 2.5, 10/RT; add 1/10 volume of 4M Tris, pH 9.0 to neutralize

-dnafactory-

:) :) Isn't there anybody who can help? :) :(

-dnafactory-

we use dss (disuccinimidyl suberate) to crosslink antibodies to protein a.

it is from a kit that we purchase from pierce (thermo), igg plus orientation kits.

we have used this kit in its previous and current incarnations for several years and have been happy with the results and stability (although, it is a little expensive).

-mdfenko-

mdfenko on Jun 17 2009, 04:42 PM said:

we use dss (disuccinimidyl suberate) to crosslink antibodies to protein a.

it is from a kit that we purchase from pierce (thermo), igg plus orientation kits.

we have used this kit in its previous and current incarnations for several years and have been happy with the results and stability (although, it is a little expensive).



Thank you so much!

-dnafactory-

mdfenko on Jun 17 2009, 07:42 AM said:

we use dss (disuccinimidyl suberate) to crosslink antibodies to protein a.

it is from a kit that we purchase from pierce (thermo), igg plus orientation kits.

we have used this kit in its previous and current incarnations for several years and have been happy with the results and stability (although, it is a little expensive).


DSS is a good crosslinker, but BS3 may be more easily used since it is readily soluble in water. You may even consider using BS2G. These high quality crosslinkers are also available from ProteoChem (www.proteochem.com) yet at much better prices than Pierce. Moreover, ProteoChem's tech support can suggest protocols to crosslink your antibody to the immobilized protein A resin.

-guru-

Dear all,
I think my problem was the starting quantity of antibody and beads. How much is your starting material? And can it be scaled down?
Thanks!!

-dnafactory-