GST or His tag for protein purification? - Tag choise + native vs denaturing conditions (Jul/14/2008 )
I have clonned a bunch of genes into the pet42a vector tagging the protein with GST, His and S tags. I could see an induction in its level after addition of IPTG with Coomassie staining. The bacteria were lysed directly in loading dye and ran on gel.
My first question is which affinity colon should I initially start out with? Using which tag can give me cleaner results? Would you recommend some brand and names of the kits for me?
Secondly, I have found Qiagen's Ni-NTA Spin kits. Could you comment on the efficiency of these kits? THey seem very easy to perform.
Thirdly, some of my genes are hydrophobic or have hydrophobic regions. Since I directly lysed in loading dye I am not sure whether using less stringent conditions during purification would cause degradation or precipitation. I see an intact band on the gel. I will use the purified protein to test immunogenecity in mice. Do I have to go for native conditions or would the denatured protein be good enough to get an immune response?
Any answer to any of the questions above is greatly appreciated!
Thanks in advance
Usually I would go the straight way with His6 purification using NiNTA column purification (gravity flow) with a Ni-NTA matrix from Qiagen (are the best) and have a look on a SDS-gel using a aliquot after purification to check for purity.
If you are OK go for proper dialysis in buffer with cartridges e.g. from calbiochem.
If not also include a GST purification step before dialysis.
However I would try to use the native protein for immunization.
To protect your protein from degradation by proteases during/after lysis I would use complete tablets (or complete minis) from Roche (without EDTA because of the Ni-NTA purification) and if your protein should be phosphorylated also PhosSTOP (also from Roche). I always use these tablets for protein stabilization during/before purification. I think at the moment you can even ask Roche for samples of the tablets if not they are not so expensive and will last for a while (and your collegues can also use them).
Good success with your experiments!
Of course do not use complete if your protein of interest is a protease
don't use PhosStop if you want to purify phosphatases
I think the spin columns will also work (easier handling) however it is more likely that impurities are eluted together with your protein than using gravity flow.