gst purification, got extra band - (Jun/05/2007 )
hi, i am doing IPTG induction in E coli. i met a problem after purification. my protein is 45kd, plus gst, whick is 26, i got a band of 71kd. but, after purification, there is still a band around 26kd. i am using the kit from amersherm for purification. i dont know what cause this extra band, could it be possible that i sonicate the bacteria too much, the protein denature and the gst showed at 26kd.
At least two possibilities:
- Your clone is not pure, as in, you did not pick a single colony but two, one with the empty vector (give you the GST band) and one true (with the fusion protein, give you the 71kD band)
- Protein degradation. This can be due to a number of reasons. Is your bacteria fresh? Induction period? Do you have PMSF and protease inhibitors in your buffer? Buffers should be keep cold, centrifugation at 4oC, sonication also at 4oC in short spurts, samples keep on ice and allow suitable resting period between sonications.
hi, i think it's protein degradation. the first time i am sonicate 10 second twice, with 10 second rest. the next time i try 10 second once, and i got more protein on 71kd and less on 26kd. now i don;t know what can i do to improve the purity, i need it to be 90% pure, now it's only 60%. and i also need the protein whichis a kinase to be active. and you know more manipunation, more easily it will lose the activity.
There are a number of options:
- If you are afraid of degradation due to sonication, can you adjust the sonicator strength level? Or otherwise, there are other ways to lyse bacteria without need for sonication, e.g. using sarcosyl/detergent method (I think PIERCE got kit for this)
- Even if your protein is not degraded, in many cases, preparation of your protein usually requires you to do dialyzation. Choose the right dialyzing membrane (since the size different between the 2 proteins is quite good), and you can get rid of the 26kD.
- How about FPLC, ion exchange....
Gentlest lysis is probably freeze-thaw with lysozyme and DNase to bust the cells and gently degrade the genomic DNA (compared to sonication which can introduce lots of variables). I think Almasy is right, however; it's more likely that you have a mixed population of cells.
If you still can't get rid of the 26 kDa using GSH columns, I'd recommend gel filtration as the final polishing step. Even if the 26 kDa protein dimerises (which GST is very good at), it'll still only be about 52 kDa, and you should be able to separate your 71 kDa protein quite simply.