GST tag protein- soluble or insoluble??? help! - (May/03/2012 )
I am trying to purify a GST-tagged protein. In the past I was able to purify some protein using the glutathione sepharose beads (after 1mM IPTG induction at 37C). However my yield was not that great so for the past two weeks I have trying different temperatures (25, 30, 37) to check if there is an increased yield. I am checking with 1ml aliquots of the culture with BPER reagent. Spin down the 1ml culture, dissolve pellet in 300ul of BPER reagent, vortex for one minute, spin down at 13K for 5 min. 25ul of supernatant taken and loaded as soluble fraction and the pellet redissolved (it doesn't completely dissolve) in 300ul BPER and vortexed. I then loaded 25ul of the partially redissolved pellet as the insoluble fraction. To my surprise in all the conditions that I tested my GST tagged protein showed up in the insoluble fraction (faint band observed in soluble fraction).
My question is this- if my protein is in the insoluble fraction it should be in the inclusion bodies. But I didn't add lysozyme to break up the inclusion bodies and my protein is present in the supernatant of the partially redissolved pellet. Is it possible that because the BPER I used is really old and doesn't extract all the soluble protein? And if my protein is insoluble how was I able to purify it in the past (NETN buffer+sonication)??? This is driving me crazzyy.......Please help! Any ideas are welcome
Check your IPTG, it may have gone off if it is an old stock.
Did you add protease inhibitors to the BPER (or did it contain them already?)? If not, it may be that your protein is being degraded.
Check your glycerol stock to make sure that the plasmid you are expressing from has not mutated.
Thanks! I prepare fresh IPTG and I can see the induction (just that it is in the insoluble fraction). I will try adding the protease inhibitors to the BPER though......