FLAG (M2) EZView beads in ChIP assay - Background binding trouble (Aug/11/2004 )
I've been working on ChIPing a FLAG epitope tagged recombinant protein that we are studying from a stable cell line we've created in our lab. So far everything else checks out at the promoter we are looking at (histone modifications, RNA pol II binding, etc.) but I have been unable to show the presence of our FLAG tagged protein. The proteins binding partner shows up at this promoter as well, so I know that I'm not looking at the wrong binding site. The problem is not a lack of signal, but that chromatin prepared from uninduced (i.e. not expressing the FLAG tagged protein) cells shows the same signal as that from induced cells.
I am using Sigma EZView FLAG M2 affinity gel beads to do my ChIPs. I know from previous experience that soluble FLAG M2 antibody will not pull down the protein. I use 20 ul of a 50:50 slurry of beads per IP in 300 ul total volume. Because the beads have a enormous binding capacity, I've tried diluting the beads 1:10 with a protein A/G sepharose mixture, as well as increasing the stringency of my washes (increasing salt concentration, etc.) in order to decrease the non-specific interaction while retaining the specific interaction.
Although epitope taged proteins are notoriously bad for ChIP, we have no other options, and I have seen publications where these exact beads are used sucessfully for ChIP (under similar conditions).
Does anyone have any suggestions or experience with this problem that they could pass on? Thanks!
I am having the same problem. I really need to do ChIP using the flag antibody but like you, I have run into pulling down lots of non-specific bands. Have you rectified your problem at all? Let me know!
Did you find a way out. I am going to be trying CHIp with FLAG tagged proteins and am worried that I will run into similar issues. 3xHA didnt work for me so FLAG is other tag I want to try.
Do let me know!
have you tried the 3X FLAG peptide elution? If not, try it and let us know your update if possible.
I wouldn't use the Flag-beads. I use those for protein purificaiton columns only. IMO they suck for coIP.
For ChIP, I've used both Flag M2 versions, IgG purified and affinity purified.
Use the affinity purified Flag M2 (Sigma cat# F1804)!! I get great signal signal with it.