Sulfo-SANPAH

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

1. Solution Preparation

(1) Working solution: Weigh the solid and prepare the required working concentration with Milli-Q water or other suitable buffer.

Note: Store solids at -20°C or -80°C protected from light. Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration yourself to explore. The working fluid must be prepared and used immediately.

2.Using Sulfo-SANPAH for extracellular matrix protein-polyacrylamide conjugation^[1] (from the literature, for reference only)

(1)In HBS buffer, dilute the selected extracellular matrix protein (e.g., collagen) to a final concentration of 100μg/mL, preparing the necessary volume of working solution.

(2) Remove all buffer from the polyacrylamide culture dish.

(3) Dilute Sulfo-SANPAH in Milli-Q water to 0.5mg/mL. Immediately use a pipette to evenly spread about 5mL of the solution over the surface of the polyacrylamide gel. Since Sulfo-SANPAH hydrolyzes rapidly in water, this step must be completed quickly.

(4) Open the culture dish lid and expose the polyacrylamide dish to 365nm ultraviolet light for 10min to activate the Sulfo-SANPAH. After treatment, the polyacrylamide should appear a deep orange color.

(5) Thoroughly wash the polyacrylamide gel three times with HBS buffer to remove any unreacted Sulfo-SANPAH.

(6) Directly add the protein solution to the surface of the polyacrylamide, ensuring the liquid completely covers the gel. Transfer the dish to a 37°C incubator, incubate overnight or for at least 2-3 hours.

(7) After incubating with the protein solution, wash once with HBS and replace with fresh HBS.

(8) Incubate the treated polyacrylamide gel in HBS at 37°C overnight or for at least 2-3 hours to stabilize the protein conjugation.

(9) The treated culture dish can be stored at 4°C, sealed with parafilm or plastic wrap for several weeks.

Note:The choice of extracellular matrix protein (fibronectin, collagen, laminin, etc.) should be based on the target cell line and tested for compatibility. Always ensure aseptic techniques are used throughout the process to prevent contamination.

References:

[1] Millar-Haskell C S, Gleghorn J P. A Large-format Polyacrylamide Gel with Controllable Matrix Mechanics for Mammalian Cell Culture and Conditioned Media Production[J]. Bio-protocol, 2023, 13(17).