I am currently using the Anti-Flag Affinity Gel to pull down a Flag protein inserted on the c-terminal of a specific protein. I have been unable to detect the flag protein via SDS page. Recently, I was able to identify the flag protein by adding DTT to the loading buffer; however, we need to pull down the flag-protein without using DTT.
Our hypothesis is that the flag tag and c-terminal is physically been blocked. What steps can we employ to unfold the protein or expose flag tag? Thus far we have only boiled the sample at 100C for 5 mins prior to SDS page.
Cheers,
If the goal is to simply detect the flag epitope (and not keep the protein in its mature form) can you not replace DTT with BME?
Your description of the issue is not clear to me. Were you able to pull down your FLAG-tagged protein with the beads but could not detect it afterwards by Western Blot? Or did you not get any signal in your pull down experiment and tested the sample directly in Western Blots this time using DTT in the SDS buffer? Was there a specific reason to use SDS-PAGE without reducing agent? If you know that your C-terminal position of the FLAG-tag might be the problem because your protein requires e.g. dimerization at that region there is no point trying to find conditions that exposes the tag which probably will change the whole structure of your complex. In that case, a pull-down experiment (assuming to find interaction partners of your protein under native conditions?) would make little sense if you change the native structure of your complexes. I would suggest trying a N-terminal tag instead. Furthermore, reducing conditions might not be healthy for the FLAG-Antibody either, so you could diminish the binding to your tag under these conditions.
It would be hard to expose the blocked, inner side peptide tag for immunocapturing. If the expression of the tagged protein is able to be changed from C-terminal to N-terminal, that would be a good idea to capture and precipitate the protein in its native form. On the other hand, DTT, TECP, or BME-like reducing agents will probably prevent the efficient binding of the FLAG-tagging and that should be the point why you are not intending to use them in your assay. ionic detergents may be used as an alternative but they can also prevent epitope interaction. Concentrations and types of reagents should be well-optimized that is highly hard stuff to implement. In this situation, you may test adding chaotropes (mainly urea) to unfold the protein and therefore expose the FLAG to affinity beads. Surely this is an option if you are interested intact but unfolded protein.
Alternatively, you may isolate your protein from the gel by extracting its denatured form. A couple of approaches are available (e.g. electroelution, passive extraction, gel dissolving). Additionally, if you find a bait protein you may convert your immunocapture assay to co-IP without FLAG tagging. Last but not least, you may think to change the tag type such as highly used Histidine, GST, Myc, Streptavidin, SUMO, Halo, etc...
Good luck, Emir
- Badges
- Science method