Agarose-conjugated antibody for IP - (Jul/27/2009 )
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!
change pH or salt concentration.
virusvirus on Jul 27 2009, 06:07 PM said:
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.
Will boiling in Laemmli buffer remove the conjugated antibody from the agarose beads?
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)