1 reply to this topic

[sixerguy98]

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]

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!!!