The protein in question may have an affinity for the crosslinked carbohydrate of which Sepharose is made. Another possibility is that the sensitivity of detecting the protein by Western blot is so high that you can detect it even thought the residual amount after 10 washes is very small.

The protein in question may have an affinity for the crosslinked carbohydrate of which Sepharose is made. Another possibility is that the sensitivity of detecting the protein by Western blot is so high that you can detect it even thought the residual amount after 10 washes is very small.

What I have learned from discussions with neighboring labs which perform co-IP experiments all the time, a big possibility is that the proteins just stick to the beads. You have to perfect the washing buffer and eluting buffer, by changing the stringency of the washes and rinses. The lab I discussed with typically wash for 1-2 hours at a time, although you can change salt concentration or heat temperature to get away with shorter time required... However, it can take a long time to optimize this procedure, and the same parameters used for 1-2 proteins will not necessarily work other proteins. There are a lot of factors to take into mind, which can effect the level of stringency required: structure and shape of protein, what types of amino acids it is comprised of, how well it interacts with other proteins (including itself), its pI, its pKa, hydrophilicity or hydrophobicity (when the protein is a membrane-crossing protein), how well the protein is folded (if expressed in-vitro, protein may not be folded as well, and may interact more non-specifically), etc..

I would recommend trying a higher salt concentration to reduce non-specific binding.

OR... in the event that the protein you are working with does contain a high amount of carboxylic amino acids, use different beads; you could try utilizing direct- pull down methods, or you can biotinylate your anti-bodies or protein, and attach streptavidin to the beads, and elute your protein this way. Another method is to use metal-coated beads- this is much more specific, however it is much more expensive. Some proteins are more of a struggle to purify than others.

The best advice:

Talk to any nearby labs which routinely perform co-IP experiments. Using a general method which was already developed by other labs, and making modifications based off the results attained from those parameters can save you a lot of time. Advice from successful labs can save you so much time, money, resources, and energy. You'd be surprised the amount of people who don't know what neighboring labs do, and the time and advice they lose from not networking with others. Most people like to help others, so it is always worth a shot.

Here are a few of the most useful websites that have helped me when trouble-shooting for co-IP:

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/co-immunoprecipitation-co-ip.html

https://www.bio-rad-antibodies.com/10-tips-for-the-western-blot-detection-of-ip-samples.html

https://assets.fishersci.com/TFS-Assets/LSG/Application-Notes/TR0064-Immunoprecipitation-guide.pdf

Best of luck!!!