I'd like to purify by immunoprecipitation a Myc-tagged protein from Arabidopsis.
I’m looking for a good kit to carry out this purpose. What do you think is the best choice, Anti-Myc affinity agarose or free anti-Myc and protein A/G? Any suggestion or comment will be welcome. Thank you so much
Nieves
In the past I have used the 9E10 mouse monoclonal antibody cross-linked to Protein G Sepharose beads for IPs and to make an immunoaffinity column to purifiy a myc-tagged protein complex from fission yeast. This was at a scale up to 5mL column volume. We were in the lucky situation where we had the c-myc cell line and so could incubate the Protein G resin with a huge excess of antibody in the culture supernatant to generate maximum degree of antibody binding very cheaply. The antibody was then cross-linked to the bead using dimethyl pimelimidate and ethanolamine / triethanolamine to quench and excess, non-crosslinked antibody removed with glycine pH2.5. We used competition with the cmyc antigenic peptide for elution of the target protein complex for silver stain gels, mass spec etc with no visible antibody leaching at all. We also used glycine pH2.5 elution for IPs, gels and blots with only very minimal leaching of the antibody from the resin. The cross-linked resin was also re-useable after cleaning with 70% EtOH. The use of the peptide will increase costs and is not really necessary for IP, but we found it useful for the really sensitive applications and to get a nice silver stain gel figure for the paper!
As mentioned above, if not cross-linking, try using the magnetic Protein G beads: quicker, easier, cleaner IPs. Also try to use non-mouse antibodies in any western blots i.e. don't use the 9E10 for primary detection as the anti-mouse secondary will pick up any leached 9E10 from the beads.
Good Luck, John
We usually use the 9E10 cmyc mouse monoclonal antibody for myc IPs. Since this is a monoclonal antibody, the IP is very specific and works very well. You can use this antibody in combination with Protein G agarose beads from any company for the IPs. Also, if you want to directly use antibody-conjugated beads for IPs, Sigma has c-myc antibody-crosslinked agarose beads. These beads also work well for performing myc IPs.
Even if myc is really difficult tag to work with, 9E10 was also in my experiment the best one.
i was working with agarose beads and it worked nicely.
Using free monoclonal antibody anti-myc and then Protein G agarose beads, the antibody is recovered with the protein of interest. Do you think this can interfere in the subsequent studies?
My objective is study the phosphorylation state of the immunoprecipitated proteins.
Thank you very much for your kind answers
That sounds like a really difficult protocol, why do you want to purify your proteins?
How do you plan to check phosphorylation of your protein?
Best regards
I presume you are going to use Western blots to detect phosphorylation, if you do, then you have to think about your antibodies traveling along if you suspend your bound material in denaturing conditions (~25 and 50-55 kDa pieces for light and heavy chains). They will be picked up by an anti-mouse secondary antibody. If your protein is of that size then you could try covalent coupling of your antibody to Protein G or elute in really low pH buffer like 2M glycine then spike that with another concentrated buffer to bring it back to physiological conditions.
Also, just my personal preference, but magnetic beads (like Protein G Dynabeads) are fantastic for IP. All you need is a magnetic stand. Super clean compared to sepharose.
My suggestion to your proteins phosphorylation status, after getting your protein purified, you can directly go for Mass spectrometry ananlysis, to study the phosphorylated/non phosphorylated status of your protein(may be after treatments you expect phosphorylation?).
I am someone new in IP and phosphorylation assays.
Ananda, yes, I expect phosphorylation after treatments.
What do you think about Pro-Q Diamond gel stain? Do you think is it a good choice to detect phosphorylation? I was thinking to use pro-Q diamond to stain directly the gel instead of doing western blot.
Thank all you for your answers and suggestions.
In the past I have used the 9E10 mouse monoclonal antibody cross-linked to Protein G Sepharose beads for IPs and to make an immunoaffinity column to purifiy a myc-tagged protein complex from fission yeast. This was at a scale up to 5mL column volume. We were in the lucky situation where we had the c-myc cell line and so could incubate the Protein G resin with a huge excess of antibody in the culture supernatant to generate maximum degree of antibody binding very cheaply. The antibody was then cross-linked to the bead using dimethyl pimelimidate and ethanolamine / triethanolamine to quench and excess, non-crosslinked antibody removed with glycine pH2.5. We used competition with the cmyc antigenic peptide for elution of the target protein complex for silver stain gels, mass spec etc with no visible antibody leaching at all. We also used glycine pH2.5 elution for IPs, gels and blots with only very minimal leaching of the antibody from the resin. The cross-linked resin was also re-useable after cleaning with 70% EtOH. The use of the peptide will increase costs and is not really necessary for IP, but we found it useful for the really sensitive applications and to get a nice silver stain gel figure for the paper!
As mentioned above, if not cross-linking, try using the magnetic Protein G beads: quicker, easier, cleaner IPs. Also try to use non-mouse antibodies in any western blots i.e. don't use the 9E10 for primary detection as the anti-mouse secondary will pick up any leached 9E10 from the beads.
Good Luck, John
We are thinking to do immunoprecipitation of GFP fusion protein. We have Arabidopsis mutants overexpressing our protein of interest fused to Myc, and another overexpressing the protein fused to GFP. What do you think? Does anybody have experience with GFP-Trap®_A Chromotek?
We are planning the experiments and we have to decide which plants we are going to use, and which IP kit.
All your answers are very useful for me.
Thank you so much
Cheers
Nieves
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