It is highly probable that the SDS flaws your assay.

I am working with prion protein, which is known for its aggregating properties.

But already 0,1 % - 0,2 % SDS eliminates any aggregation.

So yes, the detergent might mask hydrophobic interaction sites crucial for your assay (antibody binding).

Thanks Peter. Even I doubt the same.

As Peter mentioned, it is highly probable given the fact that SDS is a strong detergent with HLB value of 40, However, not all proteins are equally sensitive to SDS concentrations. Some protein-protein interactions are super sensitive to SDS while some are quite resistant. Please find the attached paper illustrating this concept. One solution to your problem is diluting the lysis buffer during immunoprecipitation. Good luck!

Tushar is right,you could try to dilute 1/10 to 0.01 % SDS and that might rescue binding.

Thanks Tushar for the suggestion. I shall keep that in mind. Do you have any idea if the interaction is between two surface receptors? I would like to see integrin-EGFR interaction.

Hi Deblina, that's a tough problem. Of course you wish to remove completely the membrane, otherwise you cannot be sure of the interaction, so milder detergents are not indicated. You have two possibilities, 1) try it with 0.1% SDS, it is the concentration used in RIPA buffer and it can work; 2) if you work on cells, do a formaldehyde cross-linking prior to lysis, this will preserve the interaction during SDS lysis and IP, but is reversible in acid, so it will revert in loading buffer (have a look at the link...).

Good luck.

http://www.hindawi.com/journals/bmri/2010/927585/

Thanks Pietro. That means 0.1% SDS helps removing the membrane from the cell also besides its role in protein denaturation, If I'm not wrong.