I 'm using pet 47b vector , i made cloning for my genes in it , i made all confirmation steps to ensure that the cloning steps( cutting, ligation, transformation) were performed accuracy and right , but when i made transformation in Bl21 DE3 and made expression at 37c for 4 hours, i haven't any difference between my inserted plasmid and negative control ( native plasmid without any insertion) in SDS-PAGE .I don't known where is the problem.
Hi dear shima.check this file. I hope it might be helpful.
https://www.neb.com/tools-and-resources/feature-articles/bypassing-common-obstacles-in-protein-expression
I agree with Michele. You should be more precise about the expression protocol. When you said that your expression construct is correct and you got colonies for the expression, the issue might be in the induction part or in the expression conditions.
Do you use the IPTG or autoinduction method? Are the expression conditions (at 37C for 4 hours) suitable for your protein? Do you express this protein for the first time or is it done before (bacterial expression systems might not be suitable for some proteins).
Sometimes when you do not get the overexpression, you might not distinguish your protein from others.
Moreover, if you are not sure about the expression of your protein, you might detect it using Western Blot or measure some kind of activity/interaction.
My protein's size is 38 KDa , and i used IPTG wiith 500 micro mole as a finial concentration is the four hours enough for the expression of such size of protein?
4 hours could be enough, but it depends. You can try taking aliquots after 4, 8 and 12 h for example. Also, you didn't mention whether you sequenced your construct. Cloning can introduce additional mutations/stop codons/frameshifts, etc., so it's important to sequence to make sure you got everything correct at DNA level.
Did you check if you are in frame?
Try to use small affinity approach to check. Is your protein toxic?
Amount of IPTG depends on the expression system you are using.
yes i am sure that my insert in frame because i used tow restriction enzymes to cut it and also to cut the vector . my protein is not toxic is cellulase.
You should try to check by a simple TPS profile after the induction protocol
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