How to improve IPTG induction? - (Mar/14/2007 )

hi
i do culture to OD600 = 0.5
To reduce the possibility of the overexpressed protein to do occlusion bodies, the induction is done at 15° for 6h.
did you put the pellet on the gel too ? i mean after lysis, spin, you may redissolve the pellet in urea buffer, eventeally sonicate and add laemmli and go for PAGE...

---

If only for CHECKING the expression of GST fusion protein by analyzing total bac lysate, I don't think you will need spin or checking pellet (you won't have pellet anyway since you just add SDS sample buffer into bacteria and run all of them). But if you don't get GST protein when you are doing PURIFICATION then yes, it is necessary to check the pellet, too. Your protein may be insoluble and stuck in the pellet. In that case, depend on your purpose, it could be a real pain in the... It may be better to do a small scale of purification of GST protein and check every step, uninduced, induced (total lysate), soluble (supernatant), insouluble (pellet), beads (bound) and elution to make sure you don't have problem or if you have, where it is, before going to bigger scale.

---

In our lab, after induction with IPTG, we incubate at temperatures like 25 oC and 18 oC depending on the protein. Also, we reduce the speed to 110 rpm, for instance, and incubate overnight.

---