Optimizing Protein Purification Conditions

3 posts / 0 new
Last post
PL11BRNDS
PL11BRNDS's picture
Optimizing Protein Purification Conditions

I am an undergraduate trying to find solution conditions to purify my protein.  My protein appears to be aggregating in solution ,meaning the aggregates are still soluble, because there is a very large absorbance peak corresponding to the void volume of gel filtration and finding the right solvent additive to stabilize the monomeric protein seems to be trial and error.  This question is more about general scientific experimentation.  What would you recommend doing if you are currently testing one combination of solution conditions or step-by-step purification method and you think of a different condition that you want to test that isn't related to the variable you are currently testing.  Here are the specifics:  I have already tested 3 different solvent additive conditions for fusion-Glutathione-S-Transferase purification as the first step.  I have also tried subsequent gel filtration for each condition.  I then tested a 4th solvent additive condition with GST purification, but substituted a second pass over the GST column in place of gel filtration and I got a purer eluate, but I have no idea if the protein is aggregating since I didn't do gel filtration.  Now I'm trying to reproduce the two-pass GST purification trial with the 4th solvent additive.  I haven't done the 2nd pass yet because I thought maybe I should try cleaving the GST tag first, then a 2nd pass of GST, and finally doing gel filtration to remove impurities.  This would require that I also test gel filtration immediately after the 1st GST pass as a CONTROL.  I think I have some good ideas, but this requires so much time that I don't have.  Please help.

protoldo
protoldo's picture
 Hola, It´s easy give

 Hola, It´s easy give recommendations that we use in our experiments  and few times run.
1.- check the isoelectric point of your protein and fusion protein and try working at some pH  as far as possible of it.
2. Try to know the prediction of solubility. For both (1,2) there are free tools in the net which giving sequence return these tips to you .
If the interactions aren´t covalents increasing ionic force of buffer, could solve it , or if they are ,probably -S-S- intermolecular adding 1-5mM Bmercaptoethanol you could excise multimers,.Other possibility is adding any detergent, if the two other methods don´t run. If the multimerization was s-s-mediated you can follow purification with no reducer SDS PAGE you mix with loading buffer without BME and load without boiling. Buena suerte, we wait for your opinions/results.

PL11BRNDS
PL11BRNDS's picture
Hi protoldo,

Hi protoldo,
Thanks for the advice.  I haven't done these things yet, but am considering them.  Using this tool (http://biotech.ou.edu/) my protein has a 52.6% chance of insolubility.  Can you recommend how I should use this info, please?  Thanks.