I have cloned a gene with N terminally fused GST in pGEX6P1. II expressed it in BL21(DE3) pLysS cells induced at 0.6 OD 600 with 1mM IPTG, 16 degree celcius overnight in 4 ml of 2YT medium supplied with AMP+Chloramphenicol+0.1% glucose. Then I prepared the Whole Cell Extract from 1 ml of culture from each sample, pelleted and boiled with 50 micro liter of 5X SDS loading dye, followed by 13,200 rpm spin for 10 minutes at 4 degree celsius and 25 microliters of supernatant from each sample was taken and loaded on 12% SDS-PAGE.
loading order as follows
pGEX6P1 Uninduced colony 1
pGEX6P1 induced colony 1-----------------GST expressed 26 kDa
pGEX6P1 Uninduced colony 2
pGEX6P1 induced colony 2 ---------------GST expressed 26 kDa
pGEX6P1-with gene, Uninduced colony 1
pGEX6P1 with gene, induced colony 1-------------GST expressed and GST fusion protein expressed (2 bands) 26kDa and 110 kDa respectively.
pGEX6P1 with gene, uninduced colony 2
pGEX6P1 with gene, induced colony 2------ only GST fusion protein expressed (single bands) 110 kDa.
Can anyone explain why the GST was not expressed in pGEX6P1 with gene colony 2 induced? In this lane I could only observe expression of fusion protein of 110 kDa but not GST.