bis-ANS (potassium salt) (Synonyms: 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic Acid)

This plan only provides a guide, please modify it to meet your specific needs.

1. Prepare bis-ANS staining solution:

(1) Prepare dye storage solution: dissolve bis-ANS in DMSO to prepare a stock solution with a concentration of 5-50mM. After aliquoting the storage solution, store it in the dark at -20 °C.

(2) Use an appropriate buffer (such as serum-free medium, HBSS or PBS) to dilute the storage solution, prepare a bis-ANS working solution with a concentration of 5-50 μM, and use a 0.22 μm filter to filter and sterilize.

Note: Please adjust the bis-ANS working fluid concentration according to the actual situation, and prepare it now.

2. Cell suspension staining (taking 6-well plate as an example)

(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.

(2) Wash the adherent cells twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.

(3) Add 1 mL of dye working solution to resuspend the cells, and incubate at room temperature in the dark for 15-30 minutes. The optimal culture time for different cells is different.

(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS to wash 2-3 times, 5 minutes each time.

(5) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through fluorescence microscopy or flow cytometry.

3. Cell adhesion staining

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, aspirate excess culture medium, add PBS to wash 1-2 times, and place the coverslip in a humid environment.

(3) Add 100 μL of dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 15-30 minutes.

(4) Aspirate away the dye working solution and wash the coverslip with PBS 2 to 3 times for 5 minutes each time.

4. bis-ANS detects changes in protein surface hydrophobicity

(1) Use 5-50 μM bis-ANS working solution to dilute the protein to a final protein concentration of 5-10 μM. Please select the appropriate dye working concentration according to specific experimental needs.

(2) Use a fluorescence spectrophotometer to record the emission spectrum at 400-600 nm, and subtract the fluorescence signal of free bis-ANS in the buffer.

5. Microscope detection: Use an optical microscope or flow cytometer to detect stained cells. The maximum excitation light of bis-ANS is 390 nm. The maximum emission light of free bis-ANS in solution is 523 nm, but after binding to protein, it undergoes a blue shift, the fluorescence intensity increases, and the maximum emission light shifts to 484-496 nm.

Precautions:

1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.

2) For your safety and health, please wear a lab coat and disposable gloves.