4 replies to this topic

[virusvirus]

Hello all:
Here's my issue (mostly my ignorance).  I have an anti-HA tag antibody conjugated to agarose beads from AbCam.  They don't have any specific information on the product, but I'm trying to use it to IP an HA-tagged protein from mammalian cell lysates.  I need to know several things:
1.  If I immunoprecipitate a protein with this anti-HA agarose, how do I get the protein off of it?  I would like to minimize the amount of antibody coming off with my protein.  Most protocols I've found indicate boiling the beads directly in Laemmli buffer.  Will this release the anti-HA antibody as well?  If it does, is there another option to avoid or minimize the anti-HA antibody being released?
2.  Does anybody have a protocol that they routinely use for IP with antibody-conjugated agarose beads?  I'm in a "collection" phase of considering multiple protocols.
3.  If anyone has ever optimized a protocol like this, can you tell me the best way to start?

Thanks everyone!  I was at the end of my rope and then remembered BioForum!

[sgt4boston]

change pH or salt concentration.

[Dr Teeth]

virusvirus, on Jul 27 2009, 06:07 PM, said:

Hello all:
Here's my issue (mostly my ignorance).  I have an anti-HA tag antibody conjugated to agarose beads from AbCam.  They don't have any specific information on the product, but I'm trying to use it to IP an HA-tagged protein from mammalian cell lysates.  I need to know several things:
1.  If I immunoprecipitate a protein with this anti-HA agarose, how do I get the protein off of it?  I would like to minimize the amount of antibody coming off with my protein.  Most protocols I've found indicate boiling the beads directly in Laemmli buffer.  Will this release the anti-HA antibody as well?  If it does, is there another option to avoid or minimize the anti-HA antibody being released?
2.  Does anybody have a protocol that they routinely use for IP with antibody-conjugated agarose beads?  I'm in a "collection" phase of considering multiple protocols.
3.  If anyone has ever optimized a protocol like this, can you tell me the best way to start?

Thanks everyone!  I was at the end of my rope and then remembered BioForum!

or elute with a competitor peptide.

[virusvirus]

Will boiling in Laemmli buffer remove the conjugated antibody from the agarose beads?

[bachai]

Boiling in LB will elute Abs that are bound to ProteinA/G agarose beads via Fc.  
Invitrogen offers CROSSLINKING Abs to prevent their co-elution with the Ag but I haven't tried it yet.
(www.invitrogen.com/crosslinking)