I want to conjugate protein having C-terminus with a dye having amine derivative through carboxyl-amine reaction. Does somebody has experience of doing this reaction? OR through EDC reaction chemistry? Thanking you in advance.
Kind Regards
The problem will be a lack of selectivity. Not only the C-terminus (if it is exposed), but also all the exposed Glu and Asp side chains will react.
If it is a relatively small polypeptide, have the peptide with label synthesized chemically by a professional service that specializes in making such things using solid-phase chemistry.
It is difficult to chemically attach a label to a single site in a large protein unless you are lucky and there is only one suitable point of attachment. The best way to achieve this is to label cysteine residues using maleimide chemistry. Most proteins have few surface-exposed Cys residues. If you want to attach the label to the C-terminus specifically, engineer the protein to replace any surface-exposed Cys residues with Ala or Ser, then change the C-terminal residue to Cys.
Another approach is to genetically engineer a fusion protein of your protein with a C-terminal fluorescent protein. Still another approach is to engineer a C-terminal tetracysteine tag that can be labeled with the FlAsH/ReAsH system:
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/high-content-screening/flash-and-reash.html
So, What should one do to get labeling of 1:1 ratio?
So use dye in excess?
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence