Hi Efraim,

Are the amount of loaded protein and the exposure time of both experiments equal? It look like trial 4 has less protein as a bait then trial 1. Maybe that can explain the lack of precipitation.

I actually don't measure the protein concentration of my pellets before preparing them for SDS-PAGE but I did load equal amounts for both of them.  And the exposure time is also the same (10 seconds). Although not enriched all the time, I always get my bait in pellet like in trial 4, usually with pulled preys. How likely it is that my IP buffer is going bad and what is the buffer you use for your IP? 

We have our IP-samples in Triton/MOPS (150 mM NaCl, 50 mM MOPS pH 7.4, 5 mM EDTA, 1 % NP40 + complete protease inhibitor). This is a rather gentle buffer.

What buffer do you use, Efraim?

Did you compare the quantity of the pelleted bait in #1 and #4? I think there is quite some difference.

My IP buffer contains;

1%NP40

50 mM tris (ph:7,4)

2.66 mM NaCl

2.7 mM KCl

1.25 mM KH2PO4

6.8 mM Na2HPO4 

Actually no, because I did pull similar amount of baits with preys in my previous IPs. That's why I didn't pay attention to it.

Hi Efraim,

I do not know whether this could solve your problem, but one issue could be the temperature. Do you performed the co-IP steps (lysis, beads washing etc...) at room temperature ? If you do, working at a cold temperature may help, and give more reproducible results, as this will help maintain weak interactions.

What are the different steps in your IP and sample preparation if I may ask ? If you use the same buffer preparations for all your experiments, maybe you should try to make it anew, and check whether it works this way.

Efram! Are you using protease inhibitor in your IP buffer as Duco pointed in his recipe.

The buffer that you use may give you some trouble. First of all the osmolarity seems too low. However, this might lead to more a-specific binding, which is not your case. Unless the #1 IP is the one with a-specific binding. Why do you use a double buffering system (tris and KH2PO4 / Na2HPO4 )

Florian, I am working on ice. In fact, in the IP step,I go to the cold room, having the samples on ice. What I do is, 

lyse the cells, take the initial sample

Use the rest for the IP. (For IP, we are using GBP (GFP binding protein) beads to pull down GFP tagged baits.  Clear lysates are tumbled for 2h in the cold room.

3 times washing with inhibitor free IP buffer.

Ritesh, I am making sure that I use the inhibitors.

Duco, I should be but I am actually not aware of the problems that my double buffer may possibly cause in my IPs. Actually the first IP trail gave me the positive control in the pellet with some unexpected fragments coming down as well? Do you think, switching buffers would help?

If you have the possibility to try the assay with a different buffer, I would encourage you to do so. I would advise you to have a osmolarity that is equal to that of the enviroment youre proteins are normally in.

The NP40 is a good choice to start with. Don't change that.

Tris is fine for most purposes, Usually it is better than phosphate-buffered solution. Phosphate can precipitate easier and may chelate with proteins. I use MOPS, actually I don't remember why we choose it over TRIS.

Protease inhibitors are a must.

Good negative controls are another requirement :)

If you see background signal (in all samples), a little higher salt concentration in the (washing)buffer might help you out.

Good Luck

Ok, I usually work in the cold room as well, and I think that one step towards reproducibility.

As has been mentionned by Duco before, the osmolarity of your sample is too low. If you try another buffer, you may wanna try with 150mM NaCl for a start. Looking at classical ripa buffers in the litterature may give you good guidelines.

Good luck !

Good comment Florian,

Do I get it right that RIPA contains SDS? This buffer may not be the one to start with. I would call it a 'rough lysis buffer'. The NP40, as used before by Efraïm, is a better one for a start. If that doesn't work, try other buffers with othe soap in it.

Thank you all very very much for putting time! I will consider all of the recommendations and hopefully be back to you with results.

Good luck!