Is it possible to label peptides with pyrilium dyes?

Yes, it is possible. Here is a step-by-step description of our experience labeling peptides with Pyrilium-1 (Py-1).

1.      1 mg of Pyrilium-1 was diluted in 100 µL of dimethylformamide.

2.      The peptide weighing 5.3 kDa was dissolved in 0.1 M bicarbonate buffer (pH 8.3) at a concentration of 4 mg/mL.

3.      3 µL of the dye solution was added to 100 µL of the peptide solution to obtain a 1:1 molar ratio of peptide to dye.

4.      The reaction mixture was gently stirred for 30 min at room temperature, observing the change in the solution from blue to red.

5.      The residue was precipitated by centrifugation at 20,000 g for 2 min.

6.      The solution was collected, and unbound Pyrylium-1 was separated by dialysis against phosphate-buffered saline (PBS, pH = 7.4) at 4 °C overnight (molecular weight cutoff = 3.5K).

7.      The labeled peptide was detected in denaturing polyacrylamide gel (SDS-PAGE), and the unlabeled peptide was loaded onto the gel as a control.

8.      Fluorescence was visualized using a ChemiDoc XRS+ imaging system.

9.      All peptides on the gel were detected by Coomassie G-250 staining for 20 min, after which the gel was washed with two hot washes in dH2O.

SDS-PAGE gel with fluorescence and Coomassie G-250 staining. (a) Fluorescence of the peptide labeled with Pyrylium-1. (b) Gel after staining with Coomassie G-250. (a, b) 1 – molecular weight marker (2-250 kDa); 2 – peptide labeled with Pyrylium-1; 3 – unlabeled peptide (5.3 kDa).