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protein expression in Arctic Express (DE3)


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#1 Me_in_a_lab

Me_in_a_lab

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Posted 20 June 2012 - 03:01 PM

Hello,
Because of the insolubility problems i came across expressing my protein of interest I decided to give it a go and try to express it in Arctic Express strain. To be honest I know little about these bacteria. I made them competent, but the manuals I have read (a fragment attached below) don't exactly say to what OD i should be growing them (24hrs in 10-13 degrees)...?
Also, is no.2 saying about "selection antibiotics" refers to antibiotics selecting for the expression plasmid, or does it mean one should not put any antibiotics at all?
Lastly, is the scale e.g. "inoculation of 1ml LB broth" just an example or this micro-scale proved to be effective while dealing with Arctic Express?

I'll be grateful for any suggestions,
regards



Preparation of Liquid Cultures Prior to Induction
1.Inoculate 1 ml aliquots of LB broth (containing 20 μg/ml gentamycin and the appropriate antibiotic for selection of the expression plasmid) with single colonies from the transformation plates. Incubate these cultures at 37°C with shaking at 220–250 rpm overnight.

Note It is prudent to test more than one colony as colony-to-colony variations in protein expression are possible.

2. The next morning, subculture the cells in 50-ml culture tubes by pipetting 60 μl of each culture into 3-ml of LB broth containing no selection antibiotics. Incubate these cultures at 30°C with shaking at 220–250 rpm for 3 hours.

Induction of Target Protein Using IPTG
3. Pipet 100 μl of each culture into a clean microcentrifuge tube and place the tubes on ice until needed for gel analysis. These will serve as the non-induced control samples.

4. Transfer the culture tubes to 10–13°C and incubate with shaking at 220–250 rpm for ~10 minutes. After the culture has equilibrated to 10–13°C, add IPTG to each tube to a final concentration of 1 mM. Incubate at 10–13°C, with shaking at 220–250 rpm, for 24 hours.

Note These values for IPTG concentration and induction time are starting values only and may require optimization depending on the gene expressed.

5. After the end of the induction period, place the cultures on ice.




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