Hi all...
I have a technical question here. Also, some fundamental doubts.
I have tagged my protein of interest with DsRED2 RFP at the C-term end. The cloning was done taking the following precaution.
1. Stop codon from the protein was removed.
2. A 6-cutter enzyme site was introduced between the protein and the RFP sequences (the 6 bp of the enzyme correspond to a Phe and Glu amino acids)
3. No IRES was incorporated between the protein and the RFP
4. However, the first codon of the RFP (Met) was left intact.
Now I have the following queries.
1. Is it possible to transfect the plasmid into HEK293 T at different concentrations and check for the expression of my protein as well as RFP by western blot and see if they are increasing at identical ratios?
2. Is there any better way to validate that the RFP tag is intact? I already see a dose response in RFP expression in microscope.
3. Is it possible that some how the ribosome can access only the RFP and it gets translated bypassing my protein since there is still Met for RFP?
What I want to confirm is that, the RFP is intact and only gets expressed with my protein and there is no premature cleavage between the protein and RFP.
Please help me understand this with your valuable suggestions.
Regards,
Sutanuka
To test whether RFP is fused to your protein of interest, use a size-specific separation technique like gel filtration or rate-zonal centrifugation. The Rf-values of the red fluorescence and your protein's activity should be identical. See for example:
@ARTICLE{Mar-61,
title = {A Method for Determining the Sedimentation Behaviour of Enzymes: Application to Protein Mixtures},
author = {R.G. Martin and B.N. Ames},
journal = {J. Biol. Chem.},
year = {1961},
pages = {1372-1379},
volume = {236},
language = {engl},
url = {http://www.jbc.org/content/236/5/1372.full.pdf+html?sid=cc0a124d-c88f-431b-90b3-3570a52e8ca2},
}
Activity of the RFP is easy to verify: it either fluoresces or it doesn't. The activity of your PoI you determine in whatever way you routinely use.
That would leave the position of attachment of the RFP to your PoI, which you can verify in a limited tryptic digestion, especially if you have sequence-specific antibodies against various parts of your PoI or if it has different tryptic cleavage sites in different conformations (i.e., after binding various ligands).
Of course, the non plus ultra would be to crystalise the fusion product and determine its structure.
The simple answer is yes, a western blot with anti-DsRFP antibody would work to verify that the RFP is attached to the polypeptide, based on the apparent MW. I believe that is the standard approach to answer your concern. Probing with antibodies against your protein in a second western to verify it runs at the same position would provide further evidence.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes