Dear all,
we need to isolate Bax protein. We have a plasmid, but when we use bacteria system (BL-21 induced with IPTG), there are low yelds of Bax because its toxicity for cells.
I think to use TnT® Quick Coupled Transcription/Translation System (Promega) for Bax isolation now.
What do you mean? Will this strategy provide better results?
Or dou you have better idea for Bax protein isolation?
Thank you!!!
Hi Petra
I recommend you to use BL21(D3)pLysS. This bacteria reduces basal expression of recombinant genes. Also you should try to induce with low IPTG concentration (< 0.5 mM)and /or low temperature (30°C).
Good Loock
Hi Petra,
to add to Farid's suggestion, if there is a risk that your plasmid has a leaky promoter, you could try adding some glucose to the medium to inhibit the promoter until you add your IPTG - this may help the cells get up to OD if they are a bit unhappy at the moment.
Which plasmid are you currently using for the Bax reading frame? Is it fused to anything?
Please feel free to discuss your question here further.
Best wishes, Robin
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