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otinanai's Content

There have been 4 items by otinanai (Search limited from 03-April 19)

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#21042 expression in minimal media

Posted by otinanai on 05 April 2009 - 03:32 PM in Protein Expression and Purification

Thank you very much...
That paper does not really solve "expression problem", more like cell growing problems... But anyway I can only thank you. I will read it thourougly tomorrow!

From a fast blink I was shocked with 8gr/L Glucose :( !!(from paper)
I thought 4gr/L was the maxium...I'll try that, but also I will try to find some more vitamins (thiamine at least).

#21011 expression in minimal media

Posted by otinanai on 05 April 2009 - 05:40 AM in Protein Expression and Purification

I'm prety sure that its expression problem and not growth! They grow as fast as they do in normal LB media, so I bet its expression issue there. I know proton-NMR is mess, and nothing special after all, but we just a first blink before we move to expensive labeled media. If we went directly to labeling we had the expression problem again plus the cost of that media!

I use Glucose for carbon source and ammonium SULFATE for nitrogen source. I dont think(and hope ) that there is any problem with sulfate, (is it?)! Yes as much as I concern M9 salts are correct. I also add trace metals BUT there is an issue with vitamin mix. By that I mean I only had Biotin :( . So Vitamin mix = just biotin... Ι found through net that there shouldnt be any problem with that. Vitamins(espessialy Thiamine) were essential in the past, modern host cells do not neccesarily require these extra vitamins.

I think I gave everything you asked , now what do you suggest? :(

#20987 expression in minimal media

Posted by otinanai on 04 April 2009 - 04:57 PM in Protein Expression and Purification

yes i want the protein for NMR. But now no I didnt label it, I just want it from minimal media for a 1D NMR first and then I will go for labeling with C and N. The protein is a U-Box protein, a part of it. tell me please what do you think, I listen!!!

#20941 expression in minimal media

Posted by otinanai on 03 April 2009 - 02:01 PM in Protein Expression and Purification

I have problem expressing my protein in minimal media. I have successfuly expressed it in LB but in the stadard M9 salt minimal media I cant. Please any help/idea is welcome. I have tried, 3hours induction(IPTG) at 37oC and overnight at 25oC but with no success.

ideas pleaseeeeeeeeeeee

P.S. host cells : BL21GoldDE3pLySs
plasmid : pet16b
tag : 6His
target protein : ~14kDa , soluble

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