I’m trying to pull down and purify GST tagged protein (target sequence is inserted in pGEX4T1 vector) from BL21 Cell lyset. Target Protein expresses in BL21 cells in good amount, confirmed by SDS PAGE. After pull-down of the GST tagged protein (5mg protein with 30ul 50% GST slurry beads), I washed the bead with 1% PBST( 5minutes for 5 times) then I eluted the protein with elution buffer (10mM reduced glutathione, 50mM Tris cl and MQ @pH 8.00). After 3 times of elution, each with a 30min incubation period, eluted samples are then subjected to run SDS PAGE ( 12% separating gel @100V ). My desire band is @ 42 kDa. In eluted protein along with my target GST fusion protein, there are some other bands (like sticky protein which binds nonspecifically with beads). I am trying to remove these non-specific/sticky proteins, as the eluted sample is followed by antibody production. I am trying with altering PBST concentration in washing step, use less volume of GST bead, and also number of washing increased to 7 times. Though, after all these changes the nonspecific bands some times vary the intensity ( target protein is with the same intensity) but not completely vanish. I'm attaching a sample copy of my SDS PAGE. If you can, give me some suggestions. Thanks in advance.
My suggestion is to follow up the affinity purification with a second chromatography step using a different principle, such as ion exchange or gel filtration, or even both. If you are going to use the protein as an antigen, you will want it to be as pure as possible, but high yield is not that important.
If you are planning to cleave off the GST tag, running the cleaved protein over a glutathione column to remove the GST may help remove the contaminants if they stick to the column, or to the GST.
An additional purification step (gel filtration) should improve the purification of your GST-fusion protein.
Thank you everyone for these valuable suggestions Im proceeding with gel filtration, will update with the same. thank you Alessandro Alaimo Yuhta Nomura Adam B Shapiro
Hello Alessandro Alaimo Adam B Shapiro Yuhta Nomura I am going through this protocol (https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/extract-proteins-polyacrylamide-gels-tech-tip.pdf). But I am worried about what is the elution buffer volume, temperature adjustment, and timing for elution. I was started my exp with 28 mg protein (before GST pulldown) and after final gel elution volume was 2ml, with 40ul elution sample, there is no protein visible in SDS PAGE. please suggest some standardized version of this protocol. Thank You.
I'm not sure about the situation. Have you performed gel filtration chromatography as a second purification step, but you are not seeing any protein on SDS-PAGE? Or, are you using polyacrylamide gel electrophoresis as the second purification step? Have you confused gel electrophoresis and gel filtration chromatography, which are two very different things?
In my opinion, purification by gel electrophoresis is not a very good method because it severely limits the amount of protein that can be purified at one time, and the recovery from electroelution is often poor. Moreover, if SDS-PAGE is used for this procedure, the protein starts off being denatured and coated with SDS.
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