Labeling Mammalian Cell Lysates (S9235)
- Harvest cells by trypsinization following established protocols.
- Wash cells twice with PBS.
- Lyse cells by suspending in reaction buffer at 104–105 cells per 20 µl. Reaction buffer is 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20, 1 mM DTT and an EDTA-free protease inhibitor cocktail (e.g., Complete™, Roche).
- Add 2 µl of the CLIP-Vista stock solution to 18 µl of cell lysate. Mix well by pipetting up and down several times.
- Incubate in the dark for 60 minutes at 37°C.
- Add an appropriate volume of concentrated SDS-PAGE sample buffer and proceed with sample preparation and SDS-PAGE according to the gel manufacturer’s instructions.
- After the gel is run, immediately obtain a fluorescent image using a laser scanner with 488 nm excitation or a UV transilluminator and an appropriate camera (Polaroid or digital). Excitation at 488 nm will give the best results. The fluorescence is an intense green.
- After fluorescent imaging, standard fixing and staining protocols can be used to detect the non-fluorescent proteins.