Dear fellow researchers, I'm having problem with DNA cloning.
I have constructed a DNA called CEP192-Myc. I tried to add Myc tag behind my protein of interest(which is CEP192). I used 'pCMV-Tag 3A' vector. I've first inserted my DNA at the front of Myc tag. Then I added additional three Alanines after CEP192 and before Myc. So, My DNA is (5'-CEP192-3A-Myc-3') as you see in the picture.
After constructing my DNA, I have transiently transfected it into 293T cells to check the proper expression. I could detect my exogenous protein by anti-CEP192 antibody, however, I couldn't detect it by anti-Myc antibody. The experiment was performed without siRNA knock-down. I could clearly see the band intensity differences between non-transfected cells(weaker bands) and overexpressed cells(stronger bands). I think that absence of stop codon after the Myc tag might have caused the problem. Maybe somewhat additional amino acids might have been translated behind the Myc tag.
My questions are,
1. Is it necessary to add stop codon after Myc tag?
2. Is it necessary to add my protein of interest solely at the MCS(multiple cloning site)?
3. Is there any tips or know-hows of making an exogenous protein with tags at the c-term(3' end)?
thank you.
Given that your sequence does have an in-frame stop codon about 27 codons after the myc tag, it probably does not matter whether you incorporate a stop codon immediately after myc or not. But you really can not be sure without trying. However it may be that your protein is being processed in the cell leaving your CEP192 domain intact but removing the myc region.
Have you sequenced your final construct to confirm that the sequence is exactly identical to what your cloning protocol would predict?
Dear Jae-Hyun,
The vector pCMV-Tag 3A is used for N-terminal tagging with myc. This means your cDNA will be cloned in the MCS, and the order will be myc-cDNA. In this case the cDNA will have the stop codon.
In the strategy you have used, you need to add the stop codon directly after the myc tag. For example see the attached image from the pCMV-Myc-C vector which is used for c-terminal myc tagging (star=stop codon)
For C-terminal tagging with myc check other vectors available from addgene.org.
There are many good commercial vectors such as pCMV-Myc-C available from Takara/clontech.
pCMV-Myc-C map is available at https://www.snapgene.com/resources/plasmid-files/?set=mammalian_expression_vectors&plasmid=pCMV-Myc-C.
Note that antibody 9E10 (Santa Cruz Biotechnology) gives a very good myc signal by western blotting.
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