In my lab we’ve been working at trying to label a protein with Cy5-maleimide for a long time. The protein has a single native cysteine residue that doesn‘t undergo the labeling reaction with any appreciable efficiency.
We have attempted an enzymatic labeling by joining a labeled peptide to our protein of interest, that also resulted in poor labeling yield.
More recently, we have used Site-directed mutagenesis to add a lysine-cysteine-lysine motif near the C terminus of the protein. the thinking was that the Neighboring lysine residues would decrease the pKa of cysteine and increase reactivity of the thiol group. Recent tests have shown that this method also has resulted in no significant labeling yield.
We are using TCEP in our reaction to reduce any disulfide bonds in our protein of interest and free up the cysteine side chains for reacting with the dye. The dye is in 5x excess over the TCEP concentration. Any suggestions would be greatly appreciated. Thank you!
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Fixatives