Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

IPTG Induction and cell lysis issues - pET 21b system (May/21/2010 )

Frustrated!!!

I have transformed my 3 proteins of interest in E-coli (pET21b system) and am having problems with any bands showing up when I do a western blot. I know that my protein might be going into inclusion bodies but would like to obtain a soluble portion to purify..just makes life easier!!!

I have sequenced and my three proteins of interest are in the "right place"..so I think my issue lies in my IPTG induction or the way I lysis my cells. My IPTG I have tried 0.2,0.4,0.6,0.8,1.0,1.2, and 1.4 mM for 3 hours at 30 Celsius. In addition, I have attempted 1.0mM at 22, 30, and 37 celsius for 4, 6, 8 hours.

For my lysis I have used sonication and also have tried B-PER Bacterial Protein Extraction Reagent (Thermo) using standary protocols. I have read before that the solution should become clear..mine stays a milkey color..any suggestions to make it clearer..could my cells not be lysised and that is why my western blot is blank?

Any suggestions would help.

Thank you in advance!!!

-sixerguy98-

Hi

I'm not sure if this help, but I've seen the same cloudy nightmare instead of a clear lysate. When I finally managed to get a clear lysate, things were fine, that is why I thought I share my way.
I vigorously followed this protocol, but I think the key issue was that I grew my bacteria at 16C overnight.

1. Day 1.
Starter culture: LB + 1% glucose + antibiotics
media in 50-ml Falcon tube inoculated
grown at 30C 300 rpm for 12-16 hrs (no longer!)
2. Day 2.
Starter culture Is kept at 4C until needed
Expression culture: media + 1% glucose + antibiotics
inoculated with 10 ml starter culture
grown at 30C 250 rpm until OD ~ 0.3-0.5 (definitely below 0.6)
3. Day2.
Induction: 1mM IPTG (this is much, less is better if yield is sufficient)
Incubated at 16C 250 rpm shaking for 12 15 hrs (I strictly kept 12 hrs)
4. Day 3.
Lysis:
Lysis buffer: Novagen BugBuster Master Mix (cat. No 71456-3 100 ml, 104)

Hope it helps,
Krisztina


sixerguy98 on May 21 2010, 04:42 PM said:

Frustrated!!!

I have transformed my 3 proteins of interest in E-coli (pET21b system) and am having problems with any bands showing up when I do a western blot. I know that my protein might be going into inclusion bodies but would like to obtain a soluble portion to purify..just makes life easier!!!

I have sequenced and my three proteins of interest are in the "right place"..so I think my issue lies in my IPTG induction or the way I lysis my cells. My IPTG I have tried 0.2,0.4,0.6,0.8,1.0,1.2, and 1.4 mM for 3 hours at 30 Celsius. In addition, I have attempted 1.0mM at 22, 30, and 37 celsius for 4, 6, 8 hours.

For my lysis I have used sonication and also have tried B-PER Bacterial Protein Extraction Reagent (Thermo) using standary protocols. I have read before that the solution should become clear..mine stays a milkey color..any suggestions to make it clearer..could my cells not be lysised and that is why my western blot is blank?

Any suggestions would help.

Thank you in advance!!!

-krisztina-