1) Have you tried a simple western blot of your two extracts with anti-FLAG antibodies, and do you get a single band in the overexpression sample and no band in the empty vector sample? Before doing IPs, you should do a western to check the complexity of your input.

2) Then regarding the IP itself, how often have you washed the magnetic beads before elution? What was the dilution factor at each step? Have you done a control with the same amount of flag M2 beads but without any protein extract, tried the elution and then the silver stain? May be the elution causes degradation of some of the antibodies on the beads. They are quite abundant and might show up on a silver stain, and yes they can degrade and give rise to multiple bands.

I'm old school and did most of my IPs with standard antibodies, followed by gravity capture with protein A sepharose, but the biological samples were in vivo labelled with 35S methionine. Radioactive Protein extracts were first centrifuged twice to remove all large aggregates. After incubation with antibodies, the samples were centrifuged again to remove unspecific antibody aggregates. The cleared supernatant was then incubated with protein A sepharose, and then gently spun at just 100 g to bring down sepharose beads only. The beads had to be washed at least 3 times, with removal of more than 95% of the supernatant to have at least a 20-fold dilution of background at each step. Then we used a fine glass needle to remove 100% of any remaining liquid and resuspended the sepharose pellet in sample buffer for boiling and loading on SDS PAGE. So if I saw the bait, and potential binders of a different molecular weight on the autoradiograph, I didn't have to worry about seeing antibody fragments on the gel, or seeing protein A fragments, because these would not be radioactively labelled and thus invisible.

If you are trying to interpret elutions from silver stains, you basically hope that your bait is more abundant than any background, and a potential binder is also abundant enough to see it in the test sample and not in the control. Also, by using flag M2 beads, you don't have the problem of protein A fragments after boiling. So the only questions remaining relate to controls I suggested above. Is there any cross-reactivity with proteins in your biological samples (Western blot of input)? Have you washed the beads enough before elution? What do you elute, and how do you elute? Can the anti-FLAG antibody itself come off from the beads (test flag M2 beads without extract)?

Thank you Jurgen for your reply.

Indeed, I did the WB against flag and I see only one band.

We washed 3 times each samples. I’ll try to test more washings. I’ll also try to elute beads only to see what is coming with just the M2 beads.

Regarding the degradation of the antibodies, I see bands higher than the heavy chain, so I guess they are contaminants.

Very soon I am going to try some protein-protein interaction work with a triple FLAG tagged membrane protein, but we have some candidate interactors that we can detect with specific antibodies....so that's a lot easier than doing silver stains. However, if we don't hit anything by educated guesses, we have to find interactors the hard way...with silver stain... Hopefully in your case more washes will sort out the background. Wishing you luck.

Hi Cédric,

Non-specific binding in IP might be due to several reasons, and you should monitor all the steps starting from your cell lysis to IP washing and elution. Start with the composition of your lysis buffer. The salt concentration is critical.

You should also check the efficiency of IP. What percentage of your target protein binds to the beads? You can easily monitor it by loading input, flow-through, and elute fractions in WB. You may then adjust your protein input or beads' volume to get good IP efficiency.

If needed, you should add a pre-clearing step, either using only beads or some other irrelevant Ab bound beads.

How long do you run IP? You may reduce the time to minimize non-specific binding. I have observed no difference in the precipitation of my target protein for 15 minutes and 2 hours.

During washing, you may increase the salt concentration in the buffer. It depends on the initial salt concentration in your lysis buffer. You can also add 2-3 steps of only water wash just before elution.

Finally, the elution buffer is most important. Elution using SDS sample buffer is very efficient. But it will also elute the Ab bound to the beads along with the non-specific proteins bound to the beads. The competitive elution using peptide may help. But the elution efficiency is comparatively low.

I hope this information might be helpful to you.

The FLAG peptide is highly poly-anionic. 3X-FLAG even more so obviously. I would suggest that the non-specific binding seen likely comes from non-specific RNA binding as RNAs and RNA fragments are also present in SDS gels. This would explain reports that single-stranded DNAs can bind with high-affinity to anti-FLAG antibodies (Lakamp et al. 2011 for instance). Has anyone tested this hypothesis directly? or found a way to efficiently and robustly circumvent such non-specific binding issues, while maintaining specific binding to the FLAG epitope?

Scott raises some good points - I will pull the thread on that myself. But, additional comments.

(1) Do you have to over-express? This is not ideal and should be avoided.

(2) Do you have to use sepharose? This resin is not especially clean. How can you be sure your "pre-clear" was comprehensive? Did you elute materials from the beads used to pre-clear? Are those bands the same as you see contaminating your IP?

(3) Are you eluting in 3xFLAG peptide or e.g. SDS -- the former will be cleaner (although yield will likely be lower, also)

(4) empty vector is not a good control - almost all antibodies have paratopes that will bind off-target in the absence of the epitope. Better off with a 'tag-only' control, or, spiking in 3xFLAG peptide to quench unbound paratopes. OR, use naive mouse IgG as the control against the cell line expressing the target - we use this control a lot, it works well for us - ideally all these controls can be done and taken together.

I have published a lot on this topic, so, perhaps the papers will be of use:

https://pubmed.ncbi.nlm.nih.gov/25757543/

https://pubmed.ncbi.nlm.nih.gov/25938370/

https://pubmed.ncbi.nlm.nih.gov/27371601/

https://pubmed.ncbi.nlm.nih.gov/28060343/

If you have questions - be in touch. I realize this is an old thread, but I have not logged in here for a while and through this may prove useful.