AF647 Picolyl Azide

Gudie (The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs)^[1]

1. Click Reaction Cocktail Assembly
1.1 Click-iT Reaction Buffer (or alkaline PBS)
1.2 Copper Sulfate (CuSO₄)
1.3 Buffer Additive/Reducing Agent (e.g., ascorbic acid or TCEP)
1.4 AF647 Picolyl Azide (diluted to working concentration, typically 2-5 µM)

2. Staining Procedures
2.1 Sample Types: Adherent cultured cells (e.g., Mia-Paca-2, HEK293-KRAS-GFP, H358, Panc-1, AsPC-1, BxPC3)
2.1.1 Adherent cells: No trypsinization is required before staining; cells are seeded and cultured in culture plates/dishes.
2.2 Incubation Conditions: Perform in situ click chemistry labeling on cells pretreated with ARS-1323 alkyne using picolyl azide Alexa Fluor 647.
2.3 Washing Steps: Wash cells with PBS after fixation and before staining.

3. Control Setup
3.1 Set up negative control: Treat cells with DMSO instead of ARS-1323 alkyne, followed by labeling with picolyl azide Alexa Fluor 647.
3.2 Set up cell line-specific controls: Include cell lines with non-G12C KRAS mutations (Panc-1, AsPC-1) and KRAS wild-type cell line (BxPC3) as controls alongside KRAS G12C-positive cell lines (Mia-Paca-2, H358).

4. Detection and Analysis
4.1 Instruments: Fluorescence microscopy system (high-throughput imaging system or confocal microscope)
4.1.1 Excitation/Emission Wavelengths: Excitation wavelength 635 nm, emission filter BA655-755 nm.
4.2 Result Analysis:
4.2.1 Fluorescence intensity change: Strong far-red fluorescence intensity is observed in KRAS G12C-positive cell lines; only extremely weak fluorescence intensity is detected in non-G12C KRAS cell lines.
4.2.2 Fluorescence localization: Far-red fluorescence localizes throughout the cytoplasm and colocalizes with KRAS protein signals.
4.2.3 Color change: This dye emits far-red fluorescence.

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