Hi,
I am having trouble with cells overexpressed mutant IDH enzyme. Problem is that it does not work. In other words, I chose some genes as a positive control from other papers and evaluated their mRNA expression levels, but there is no difference among cells carry IDH wt, IDH mutant and vector plasmid, unlike in the other papers. I checked the construct through sequencing again, construct is correct. Cells are at passage 12 and they have still protein expression ( checked by WB), Cells were infected by using retrovirus. I increased the concentration of L-Glutamine in culture media to boost the level of a-ketoglutarate level because this mutant enzyme needs a-ketoglutarate to produce 2-hydroxyglutarate. Cells are glioblastoma cell lines and media: DMEM High Glucose media. If you had encountered like this problem before and share your experiences and knowledge with me, I will appreciate.
Thanks,
Hi, it is not clear what your experimental problem is. You seem to suggest that you are detecting protein expression of the mutant enzyme by western blot and potentially mRNA. Is this correct?
Hi Erik,
I wonder are there other people who have problems with that protein or not specifically having a similar issue about another protein, how do you check whether a protein works well (functional) except construct checking, some targets' expression (indirectly) (as a positive control) or enzymatic assays. I may miss some points. Therefore, do you have any suggestion?
Thanks
I'm still confused as to what problem you are having.
Have you checked you expression after transfection for both the wt transfected and your mutant by qPCR and western blot? How are you differentiating between endogenous IDH and your transfected? If you have no way of doing so, you might want to tag your mutant and your wt that are in your plasmids. This will at least let you know you have expression from your plasmids. Then, you will be able to focus on the function/activity of the mutant enzyme. If you already have the cells stably transfected, you could knock down the endogenous IDH enzyme with siRNA that only targets the endogenous. Alternatively, you could design PCR primers that only amplify your transfected wt and mutant enzyme, but not the endogenous. Until you unambigiously demonstrate you have expression of your mutant and wt transfected IDH enzyme, it will be very difficult to do further investigations into the activity of the mutant.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques