Protein preicipitated after Dialysis - (Nov/18/2008 )

Hello Everyone!
I am trying to purify my protein of different sizes with almost equal pI ranging from 5.98 to 6.11. After doing Co column purification, the OD comes out to be fairly good but when I do the dialysis to take out the salts from my protein, my protein get precipitated out. I use 25mM Tris ph 7.8 to dialyse the protein. I am keeping the dialysis bag at 4deg but still it gets precipitated. I am not able to figure out what should I do next. AFter dialysis, I do the HTQ column but the protein quality decreases.. I mean that I am not getting good protein out of it.

Kindly help me.

Thanks
Khushi

Screen for different buffers at different concentrations and different pH. Proteins sometimes are more happy in one buffer than in another (even at the same pH). You can also try to increase the concentration of other salts (NaCl) in your dialysis buffer.

What buffer is the protein in before dialysis (elution buffer)?

The protein I work with precipitates in Tris, Sodium acetate, phosphate buffers and anything with salt. But MOPs and MES work fine!

I have had this same problem with several proteins. Firstly, if your protein is too much concentrated it is easier to agregate, try to dilute the sample, you can always concentrate it afterwards by using a IEX resine for instance which have quite big capacity. Sometimes adding several additives helps to avoid agreggation: NaCl 150 mM, trehalose 15%, emulfogen 0.1% or triton 0.1%. The pH you want to reach is very important, sometimes you can have agregation because it is nearly the pI of the protein and sometimes because the proteins does not like this pH, who knows why......, Do you really need to use this specific pH?, may be to bind subsequently to another resine?, if not you can find out a different pH more suitable to your protein. I don´t know if you need that you protein was completelly functional, if not, you can use some caothropic agents such us urea 4-6 M or so. I know that sometimes aminoacids such us arginine are useful to avoid agregation.

Sorry!, what is HTQ column?. If I am correct, in a Co column you are using at least pH 8, am I right?, and the protein was ok in this pH?. I do not understand the hole procedure, could you explain it in more detail regarding resin, buffer and pH in your purification procedure?. You just want the dyalisis to get ride of the salt or to change also the pH?, because maybe in the elution from the Co affinity column you could elute without salt at a conductivity in which you can bind directly your protein on the next resine by-passing the dyalisis step.