I have no tags for the gene that I have cloned in pCDNA. If I have to detect the success of transformation, it can be detected by geneticin selection. Does it also imply that the gene is expressing the desired protein?
Not all cDNAs get successfully transcribed into mRNA, not all mRNAs will lead to protein production. Make sure you've sequenced your cloned cDNA so that you can eliminate the possibility of mutations. Use immunoblots and/or immunofluorescence to confirm that your GOI is expressed. If this gene is expressed endogenously, you'll need to demonstrate that the levels are increased after transfection or find a cell type that lacks your GOI for expression studies.
Eventhough you may not need a tag to prove that your transgene is present, it is not safe to assume that just because a cell is geneticin-resistant that it is also expressing your GOI.
Not all cDNAs get successfully transcribed into mRNA, not all mRNAs will lead to protein production. Make sure you've sequenced your cloned cDNA so that you can eliminate the possibility of mutations. Use immunoblots and/or immunofluorescence to confirm that your GOI is expressed. If this gene is expressed endogenously, you'll need to demonstrate that the levels are increased after transfection or find a cell type that lacks your GOI for expression studies.
Eventhough you may not need a tag to prove that your transgene is present, it is not safe to assume that just because a cell is geneticin-resistant that it is also expressing your GOI.
Dear Daniel,
Thanks for your valuable help. I completely agree with you. The problem here as i mentioned that i do not have any tag with my GOI so the detection problem arises. As you rightly suggested other techniques to detect are immunoblots or immunofluorescence. Can you suggest any other technique which can be used for the detection apart from immuno-studies?
I thought it would be really good to have GFP tag or His tag for that matter to get easy access to MAb. It would be really great if you could put some light on this thought.
Amrutraj- you'll need an antibody directed against your gene of interest. If such an antibody does not exist or cannot be acquired, you have to use an epitope tag instead. I wouldn't recommend a His-tag for mammalian cell expression; GFP is good for live cell imaging, but for purposes of immunoprecipitation or Western blots, a FLAG, Myc, or HA tag are most commonly used. I'd recommend introducing a tag into your pCDNA construct if you can (make sure the tag and cDNA are in the same reading frame).
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