Hi,

I would consider using some unrelated purified proteins which are not expected to interact with your target protein (bacterial proteins if your target is from eukaryotic sources for example).

Then you could estimate - on average - which conditions are stringent enough to get rid of the non specific binding with the chosen "standard" proteins.

Anyway the boundary between specific and non-specific interactions may be difficult to establish exactly. Some specific interacrions may be weaker than non specific ones but have a biological effect (if they induce a conformational change of an active site for example).

Max.

In context of Co-IP and GST pull down assay include a control reaction with one of the interacting partner missing.

I suggest an NaCl gradient and independently a Triton X-100 gradient. There is no right or wrong strength, only at this point documentation compared to physiology. Based on these findings you can add albumen and other cellular components.

I agree with Amit that control is very important in any stringent washing conditions. My suggestion is that you can first try to identify strong binding proteins using most stringent condition such as 500mM NaCl in your washing buffer, and than work on weak binding proteins. For some very weak binding proteins, you may need add cross-link reagent such as DSP.

thanks for the infor..very much appreciated..

by the way, as mentioned by Max, 'Some specific interacrions may be weaker than non specific ones but have a biological effect (if they induce a conformational change of an active site for example)', if this is the case, then how can we identify the interaction.?

Specific interaction are usually stronger

The most meaningful in vitro conditions are likely those producing the results closest to the in vivo conditions.

I would start with high stringency first as Chunlei suggested and then move to the weaker interactions. In this latter case you could verify if the in vitro observed interactions occurr also in vivo by setting up an in vivo assay like the yeast two hybrid system (simple animation at http://www.youtube.com/watch?v=vk3I4xldC7o).

This could be used to verify the results of your in vitro conditions.

The in vivo assay would be a benchmark to calibrate the right buffer stringency at least for the "borderline" interactions. After this you could just work in vitro assuming that the interactions that you detect will likely have a biological activity within the cell.