I´ve been trying for a long time to express a cytoplasmic tail of a transmembrane protein and purify it. Since the beginning I have experienced several problems with inclusion bodies. I was able o purify the inclusion bodies and then solubilizing them using 8M Urea, and it worked successively! However, it seems that the protein is coming out of solution after the purification (in the eluate). Since I can not change my elution buffer, do any one of you have any idea how can I solve this problem?
It sounds as though it is just very insoluble or that in high concentrations it may aggregate and precipitate. This paper may help:
J Am Chem Soc. 2004 Jul 28;126(29):8933-9.
A simple method for improving protein solubility and long-term stability.
Incubation in Tritonx-100 for at least 10 minute will rectify the error to a certain limit.
I have tried this protocol from Cold Spring Harbor, it works well. Good luck!
http://cshprotocols.cshlp.org/content/2009/11/pdb.prot4997.long
Chao-Fan Ji may right, I also did the same in my work on Acid-phospahtase enzyme variation in the brain of female mice, the result was excellent.
try with a sequential dilution by reducing the strenght of the buffer and the suggested Triton trick.
I think using urea for protein solubilization is not a good idea. Urea denaturates your protein and avoids the binding of the GST tag to the affinity column used for purification. I suggest the use of Triton in combination with other detergents such as Sarkosyl and CHAPS for protein solubilization before affinity cromatography. An article that could help you is attached. Good luck!!!
I use 8 M urea followed by a 42 degrees overnight step with 10 mg/ml n-octyl-b-D-glucopyranoside (OGP) detergent. Then I dialyse it out (agaisnt the buffer +0.01% OGP) before running it through the GST column.
Don't run a column fast. Do a native gel low percentage. Identify your band using antibody (against GST) on gel. Use high concentration of Ab in a little buffer. Don't allow to diffuse your band do it in RT for 1 h. Cut your band extract it with buffer. Run your column. Elute your column with week buffer fast (to get read of contaminant), then elute it with concentrated buffer (to get your protein). Once you get pure protein then try to convert it in active form. Best of luck.
I would try and avoid urea as your protein is solubale until you elute from the column. Urea bringgs its own problems including confirming you have refolded the protein in an active form. I would first work out if it is a concentration problem maybe collect the eluate in a vial containing a specific volumn of elution buffer ie if you elute in 1ml frations have 1 or 2 mls of eluation buffer in the tube already therby dilutuing the protein during elution. However bare in mind that the protein may have reached this critcal conc on the column so it maybe wise to increase the bed volumn. If you are going to try a detergent start with CHAPS as this can be removed downstream. Using any of the Triton series bring there own issues least of which they cant be removed once they are there.
Try using a mixed micelle of 1.5% sarcosyl and 1.5% triton x-100 to isolate proteins.
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