Hello all, first post on ResearchGate!
I have been performing immunoprecipitations with FLAG beads (abcam, ab270704), eluting using SDS loading buffer (with DTT) and boiling. The eluted products are then used for SDS-PAGE and downstream Western Blots. We're concerned our proteins of interest are not being fully eluted from the beads using our current method. Abcam suggests a competitive elution using the DYKDDDDK peptide, however I'm having trouble finding it for purchase from them. Has anyone used that peptide product from another company and had success?
Thanks!
If you want to elute your protein from the beads under native conditions, then the FLAG peptide is probably a good way to do it. If you are going to run the protein on an SDS-PAGE gel, then eluting with SDS and DTT and heating should get the job done, since that should denature the anti-FLAG antibody. I doubt that adding the FLAG peptide will enhance the elution any further.
What makes you think the proteins of interest are not being fully eluted?
Adam B Shapiro is almost definitely correct in advising that competitive elution will prove less effective than boiling SDS.
Theoretically, elution with peptide will be more specific, so you'll elute less of the inevitable non-specifically bound protein. But depending on your binding conditions, some amount of your target protein is also non-specifically bound, even though it is capable of specific binding. And competitive elution is highly unlikely to elute that non-competitively bound material. So the bottom line is, with peptide elution your yield is unlikely to increase over boiling your beads in SDS.
What makes you think the proteins of interest are not being fully eluted?
Hello all, thanks for answering! The reason we think our beads are perhaps not being fully eluted, is basically because our co-IP's are coming up empty. So we are pulling down plasmid-based POLE1-Myc-DDK, and blotting for POLE1 alone with several other replication associated proteins (POLE2, POLE4, PCNA, MCM2, etc). We are seeing bands (as expected) in the POLE1 blots (which we want to check for IP verification), but we're not seeing any other proteins come down with it, which doesn't make sense. The antibodies are able to detect the endogenous replication factors I mentioned, but not in the IP. Also even when POLE1 is pulled down, it seems like there's less of it than there should be (although I don't have a formal quantification of that). So we were wondering if the elution of the beads was incomplete, or if it's another issue entirely.
Ah! Westerns are complex assays, susceptible to many problems. Let's back up your experiment and run some controls.
1. What makes you confident your detection Ab are able to detect your target replication associated proteins after you blot them from a gel you run with POLE2, POLE4, PCNA, MCM2, etc. standards? Does your Ab provider attest to their utility in Western? Many Ab work in ELISA and histo but not in blots, because the antigens are more denatured by blotting.
2. What makes you confident your probe (2nd Ab) lights up your detector Ab? Assuming your detector Ab are mouse monoclonals, you need an anti-mouse probe. Then, whether the probe is specific for the heavy or light chain of the detector Ab, make sure it matches the detector Ab type.
3. Check your probe chemistry.
You can check both 2 and 3 with a dot blot. Spot 1-10 ug detector Ab, onto a clean Western membrane (for #2). Spot probe Ab as a control (#3). Then wash and develop as usual.
4. Are you certain you express the target proteins in your experimental model? Are you using a protease inhibitor in your lysate?
You might run the experiment without the pull-down enrichment step. Then overload your gel (use neat lysate) and blot for your targets. Your bands will be ugly, but if you don't see them at all, and you're sure of #1, 2, and 3 (above), then maybe #4 is not working.
So our proteins of interest (replication factors I mentioned) are showing up in the input samples, so that shows our antibodies are working (sorry, I should have clarified that earlier). So that's why we're zeroing in on our IP has having some sort of issue.
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