Sulfo-cy3 picolyl azide

Operating Instructions (The following is a recommended experimental protocol for guidance only; adjustments should be made according to your specific requirements)
1. Stock Solution Preparation
1.1 Solvents: H₂O, DMSO; alternative solvents can be selected based on experimental needs.
1.2 Concentration recommendation: 2 mM.
2. Working Solution Preparation
2.1 Diluents: H₂O, reaction mixture containing CuSO₄, THPTA and sodium ascorbate.
2.2 Working concentration: 100 μM, 2.00 μmol per click reaction.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh before use.
3. Staining Procedures
3.1 Sample type: Immunoprecipitated protein complexes (HA/antibody/Protein-G-sepharose)^[1]
3.1.1 Incubation conditions: Use 100 μM Sulfo-cy3 picolyl azide working solution; incubate at room temperature for 1 h; protect from light.
3.1.2 Washing step: Wash with PBS at least 4 times.
3.2 Sample type: Purified recombinant protein samples (DDB1, DDB1-CRBN)^[2]
3.2.1 Incubation conditions: Incubate with Sulfo-cy3 picolyl azide solution; perform UV crosslinking after incubation.
3.3 Sample type: Adherent mammalian cells (MCF7, HEK-293)^[3]
3.3.1 Incubation conditions: Incubate cell lysate with 2.00 μmol Sulfo-cy3 picolyl azide at 4 °C overnight; prior to staining, cells need to be treated with probe and exposed to UV light, followed by cell lysis.
4. Control Setup
4.1 DMSO control: Rule out solvent interference.
4.2 CRF (control reference fragment) control: Verify the specific interaction between the fragment and protein.
5. Detection and Analysis
5.1 Instrument types: Biomolecular imager, gel imaging system for fluorescence detection, fluorescence imaging system, in-gel fluorescence scanner.
5.2 Excitation/emission wavelengths: Excitation wavelength = ~550 nm, emission wavelengths = 560 nm, ~570 nm.
5.3 Result analysis
5.3.1 Fluorescence property: This dye emits red fluorescence.
5.3.2 Signal intensity correlation: The higher the molar ratio of alkyne to azide, the stronger the fluorescence signal; an enhanced fluorescence signal indicates direct binding between the target fragment and protein; a decrease in fluorescence intensity may be observed under specific experimental conditions.
5.3.3 Fluorescence localization: The signal corresponds to the target protein (HA) separated by SDS-PAGE; the localizes to the protein bands corresponding to DDB1 or DDB1-CRBN on the gel; in MCF7 cells, the signal colocalizes with the endoplasmic reticulum marker calnexin.

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