Hello everyone,
I'm quitte at a loss here. I'm using the DYKDDDDK Tag Monoclonal Antibody (FG4R) (Catalog # MA1-91878) from thermofisher and while it works beautifully when I blot against Flag or use it to pull-down Flag tagged proteins. I can't see any signal when I use the same antibody to blot for Flag after the IP. If I use an antibody against the protein itself and not the tag, I have a beautiful signal and the shift due to the tag is clearly visible. When I use the Flag antibody, I have no signal.
Did anyone experience that issue ?
Thank you in advance for your help :)
Wilhelm Vaysse--Zinkhöfer
so you've done controls with the (non IP'd) original protein on the same blot?
@Edward yes ! As long as I didnt perform an IP first I can see the flag-tag. Tried rabbit and mice flag antibodies and protein A.. Im quite at a loss.
How do you elute the FLAG-tagged protein from IP beads (If applied)? Are you able to confirm the tag is still fused to the protein after the IP cycle by employing mass spec analysis? If I understood correctly, you are cleaving off the tag from your POI after the IP application.
İsmail Emir Akyildiz The flag-tagged protein is co-ipped with a HA-tagged one (which works perfectly). I confirm the presence of the tag by the shift of the protein compared to the endogeneous one. I might be cleaving the part of the peptide the antibody targets ?
I might be cleaving the part of the peptide the antibody targets?
This can only be confirmed by mass spec analysis (PMF analysis)...
Shifted transition is alone not a reliable way to assess whether the FLAG tag or peptide attached was removed or remained intact. For the peptide cleavage confirmation, bottom-up proteomic is needed to conclude...
How about the conformational (high-order structural alterations) modifications after IP elution which results in the FLAG being stuck into the conserved region? Is it possible to become a steric hindrance for the primary antibody to be bound to the FLAG tag after the IP treatment?
Here are a couple of additional considerations;
1)You may modify the construct and switch the position of the tags. Next, observe the results following the same assay sequence (IP and WB tandemly). This may solve the issue without performing any troubleshooting approaches.
2)Perform experiments in comparison using all denatured and non-denatured conditions to get some hints if the structural variations can affect your experimental outputs.
Emir...