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[Janesh Gautam]

HII FREINDS

I HAVE CLONED MY FZR1 cDNA  INTO BACTERIAL EXPRESSION VECTOR pET 29 a +. FZR1 IS A HUMAN PROTEIN . I AM TRYING TO EXPRESS IT IN BL21 DE3 & BL21 pLYSE BACTERIAL STRAING .I AM DOING IT AT 37DEGREE AND INDUCING IT AT 0.3 mM IPTG . MY GUIDE IS TELLING ME TO SEARCH THR LITERATURE FOR ITS INCUBATION TEMPERATURE . I AM NOT GETTING IT .

PLZZZZZZZZZZ HELP ME

[protolder]

Janesh Gautam, on 03 April 2011 - 09:54 PM, said:

HII FREINDS

I HAVE CLONED MY FZR1 cDNA  INTO BACTERIAL EXPRESSION VECTOR pET 29 a +. FZR1 IS A HUMAN PROTEIN . I AM TRYING TO EXPRESS IT IN BL21 DE3 & BL21 pLYSE BACTERIAL STRAING .I AM DOING IT AT 37DEGREE AND INDUCING IT AT 0.3 mM IPTG . MY GUIDE IS TELLING ME TO SEARCH THR LITERATURE FOR ITS INCUBATION TEMPERATURE . I AM NOT GETTING IT .

PLZZZZZZZZZZ HELP ME

Hola, relationship between induction and expession depends of each protein, but as its mol.weight increase more possibilities of find the induced proteins as insoluble inclusion bodies. for me, always would rather little amount of soluble protein, to high one insoluble that you have denature and refold, Your protein seems of the complicated ones so try induce with this concentration of IPTG (in LB medium at 0.5-1 units of OD at 600nm)lowerig temperature at 28ºC with 3 hours of induction. Analyze expression and at 1-2-3h and vary inducer concentration or temperature as the results indicates to you. Buena suerte