Trouble in GST- fusion protein purification - (Mar/10/2013 )
I have been working to purify a GST- fusion protein (62KDa) which is in BL-21 vector.my protein is expressing well, but i couldnot get the protein from purification using the kit Genei . I have been using the protocol ...,
overnight culture - 3ml in 250ml LB broth; in shaker 37c ,160rpm untill OD - 0.6 - 0.7(TAKES 3-4HRS ) - 0.3mM IPTG IS added and allowed fro 4 hrs.
pelleted out -12000rpm ,5mins,4c and left in - 70c.
below steps on ice
nextday- to pellet 15ml of equilibration buffer provided with kit is added and homogeneited ,100mM PMSF IS added , sonicated for 3osecs brusts with same time on ice repeated for 20 cycles. the supernatant looks clear..,
solution A ( TRITION 1%) 0.5ml added immediately and inverted left for 30 mins.
14000 rpm for 30mins and ,column eqilibrated with 10ml of equilibration buffer supernatant to the column ( agarose column of 1ml) -repeated for 4 times
and washed with same equilibration buffer till OD of 0.05 in 280nm ;elution buffer 500microliter added eluted again to column repeated for 4 times . but no protein in three elutes.
How are you checking for the presence of protein? Through your tag or POI?