Dear All: Thanks a lot for the prompt replies. The following are the good ideas I have got so far besides some details that I forgot to mention earlier.
I tried 10 buffers from pH5 to pH9.0 at 5mM with a final protein concentration of 0.5mg/ml to 1mg/ml. And I also tried to add 0.1M NaCl at pH7.0 or pH7.5. I still can see the precipitates. Actually I got one clear mixture at pH4.6 but I am not sure whether these two proteins will interact with each other at this point. Welcome new ideas. Jerry 1) Anastassis Perrakis Try mixing at lower conentrations (100 times or so) and concentrate them afterwards 2) Juergen BoschWhat was your buffer and how much ? What protein concentrations in mM did you add ? Did you have salts present ? They could shield the high pI protein or they could be removed from your other protein and attracted to the high pI protein, which then crashes out ? 3) Stephen GrahamI had the same problem once. It was a simple matter of screening a number of different buffers (at different pHs) until I found one where proteins were stable in complex. In my particular case I had to drop the pH below 6 then the proteins were eminently happy together. You can easily screen a multitude of buffers by adding a small amount of the two concentrated proteins to a larger volume of buffer, waiting 10 min, spinning down the sample and then looking for a drop in [protein] (using the A280, bradford assay, etc). 4) Pius Padayatti you ca try changing the buffer to another if you know under what conditions you could determine the binding affinity. Also try adding DTT etc in fresh to prevent any unwanted reactions. Just a simple suggestion hope it helps 5) Jeremy Tame There was a nice piece in Nature about 10 years ago showing that if many polystyrene balls of two sizes were mixed and allowed to diffuse around the surface of water, beyond a given concnetration the larger ones become close packed at the sides to allow the smaller ones more room. It's a pure entropy effect. In your case I suspect the pI may be playing a role, though you don't mention the pH of your buffer. I would try expressing the two proteins as a single polypeptide. That way they aren't going to let go easily! > From: [EMAIL PROTECTED] > Subject: Re: [ccp4bb] FW: protein induced precipitation of proteins? > Date: Tue, 4 Dec 2007 10:38:36 +0900 > To: [EMAIL PROTECTED] > > Hi Jerry > > There was a nice piece in Nature about 10 years ago showing that if > many polystyrene balls > of two sizes were mixed and allowed to diffuse around the surface of > water, beyond a > given concnetration the larger ones become close packed at the sides to > allow the > smaller ones more room. It's a pure entropy effect. In your case I > suspect the pI may > be playing a role, though you don't mention the pH of your buffer. I > would try expressing > the two proteins as a single polypeptide. That way they aren't going > to let go easily! > > good luck > Jeremy Tame > > > > > On Dec 4, 2007, at 2:28 AM, Jerry McCully wrote: > > > sorry for the repeats. > > Jerry > > From: [EMAIL PROTECTED] > > To: [EMAIL PROTECTED] > > Subject: protein induced precipitation of proteins? > > Date: Mon, 3 Dec 2007 09:25:51 -0800 > > > > Dear All, > > > > Recently I was trying to crystallize a complex of two > > proteins(40kD vs 20kD). TO my surprise, precipitates immediately > > appeared in the solution even when I mixed these two proteins at about > > 1mg/ml. However, the individual proteins were just soluble and stable > > in the same buffer. From the binding studies I knew that the affinity > > between them was lower than 1uM therefore I could not purify the > > complex by gel-filtration. > > > > I checked the precipitates by SDS-gel. The majority of the > > precipitates were the bigger protein. I'd like to mention that the > > smaller one was a protein with the pI above 10. > > > > Did the smaller protein induce the precipitation of the bigger one? > > > > Does anybody know some tricks to avoid the precipitation so that > > the protein complex could be concentrated enough for the > > crystallization trials? > > > > Many thanks in advance. > > > > Jerry McCully > > > > > > > > Your smile counts. The more smiles you share, the more we donate. Join > > in! > Share life as it happens with the new Windows Live. Share now! _________________________________________________________________ Share life as it happens with the new Windows Live.Download today it's FREE! http://www.windowslive.com/share.html?ocid=TXT_TAGLM_Wave2_sharelife_112007
