2.6. Optimization of sortase-mediated fluorescent labeling

Sortase labeling of proteins requires optimization of three variables: sortase variants, temperature, and time.

Prepare two master mixes with a total volume of 160 μL containing either the sortase (5M) or sortase (7M). Each master mix includes:

Protein to be labeled (1–20 μM is recommended).

3–5 × sortase compared to the concentration of the protein to be labeled.

5 × fluorescent peptide compared the concentration of the protein to be labeled.

10 mM CaCl₂ for the master mix containing the sortase 5M variant.

Divide each master mix into 15 separate aliquots of 10 μL and incubate at the specific time and temperature.

Test sortase labeling at 4°C, 15°C, and 37°C. Also, test different incubation times (i.e., 0.5, 1, 4, 6, and 12 h).

Quench the reaction by flash freezing the aliquot in liquid nitrogen and storing at —20°C until all reactions are complete.

Run all reactions on an SDS-PAGE gel and acquire a fluorescent image of the gel using a Typhoon.

Stain the gel with Coomassie to determine the protein concentration.

Calculate the ratio of fluorescence intensity in the Typhoon image to protein concentration in the Coomassie-stained image for each condition to determine the best labeling condition.