AF488 Picolyl Azide

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation

2. Working Solution Preparation
2.1 Diluent: PBS.
2.2 Working concentration: 1 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For adherent cells (U2OS cells)[1]:
3.1.1 Seed cells directly in culture chambers; no trypsinization required.
3.1.2 Incubation conditions: Incubate with 1 μM AF488 Picolyl Azide-containing click reaction mix for 1 hour in the dark.
3.1.3 Washing steps: Wash 1 time with 3% BSA in PBS for 5 minutes, 2 times with 0.5% Triton-X in PBS for 10 minutes each, and 2 times with PBS for 10 minutes each.
3.2 For suspension bacterial cells (Bacillus subtilis)[2]:
3.2.1 Incubation conditions: Use AF488 Picolyl Azide for CuAAC reaction with metabolically alkyne-labeled cells.

4. Controls
4.1 Negative control: Cells treated with vehicle (DMF) only, without Pt-alkyne-53.
4.2 Probe-only control: Cells treated with alkynylated DMAPP analog (1) without Cu(I) catalyst.
4.3 Catalyst-only control: Cells treated with Cu(I) catalyst without alkynylated DMAPP analog (1).
4.4 Competition control: Cells co-treated with alkynylated DMAPP analog (1) and 10 μM SIE-DMAPP.

5. Detection & Analysis
5.1 Instrument type: Confocal microscope.
5.2 Ex/Em wavelengths: Corresponding to Alexa Fluor 488 (green fluorescence).
5.3 Result analysis:
5.3.1 For adherent cells[1]: Green fluorescence is detected in target-treated cells, with no green fluorescence in vehicle-only control cells; fluorescence localizes primarily in the nucleus and nucleoli, with additional detection in the cytoplasm, appearing as green signal against blue DAPI-stained nuclei.
5.3.2 For suspension bacterial cells[2]: Green AF488 fluorescence is primarily confined to the cell periphery (membrane) and enhanced at cell division septa; fluorescence intensity is reduced in competition controls with SIE-DMAPP supplementation, and no fluorescence is detected in probe-only or catalyst-only controls.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.