Hello every one, I have been inserted Metallothionein gene and lead binding poly peptide into SB1C3 vector and transformed resulted plasmid in E. coli DH5a. How I can measure the gene expression or amount of Mt and PbBP proteins?
For protein expression, you could use Western blot methods if appropriate translation to protein has been attained. As far as protein activity - you could sieve-column purify for proteins of appropriate MW and then assay for enzyme kinetics using appropriate substrate(s), co-factors and associated spec. measurements of the ensuing reactions using Michaelis-Menten based analyses. And, if you've achieved appropriate mRNA expression (that can be linearly correlated with protein expression) you can use qPCR for that. Hoping that prokaryotic machinery can faithfully translate eukaryotic protein would have to be predicated on the appropriate use of prokaryotic Shine-Delgarno in lieu of eukaryotic Kozak sequences at the point of translation, etc. You may want to explore mammalian expression vector designs as well...
For protein expression, you could use Western blot methods if appropriate translation to protein has been attained. As far as protein activity - you could sieve-column purify for proteins of appropriate MW and then assay for enzyme kinetics using appropriate substrate(s), co-factors and associated spec. measurements of the ensuing reactions using Michaelis-Menten based analyses. And, if you've achieved appropriate mRNA expression (that can be linearly correlated with protein expression) you can use qPCR for that. Hoping that prokaryotic machinery can faithfully translate eukaryotic protein would have to be predicated on the appropriate use of prokaryotic Shine-Delgarno in lieu of eukaryotic Kozak sequences at the point of translation, etc. You may want to explore mammalian expression vector designs as well...
Dear Jack M Gallup Thank you very much for all of these information, its seems some new methods for me but I 'll look for its protocols. Thank you :)
An ELISA might be more quantitative than a western blot, if you need more then a "yes/no" answer for the expression. You could also do a dot-blot to screen your transfections.
Hello,
Following on Jack's suggestion. If you would be performing a western blot, don't forget to compare it to a "proper" control. You would want the expressed protein (in E.Coli) to have the same migration as the endogenously expressed protein. If this is not the case this can show various alterations (relative to the desired ) structure of the protein including incomplete / different PTMs such as phosphorylation / glycosylation or extensive / incomplete processing.
Also don't forget to consider alternative expression systems such as yeast, insect and of course mammalian.
Good luck!
For gene expression you may very well go for qPCR where you can quantify the expression of metallothionein and its various isoforms to be more specific.western blot can help in quantifying the protein expression
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