Home Protocols Labeling Mammalian Cell Lysates (S9235)

Labeling Mammalian Cell Lysates (S9235)

Protocol

  1. Harvest cells by trypsinization following established protocols.
  2. Wash cells twice with PBS.
  3. 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).
  4. Add 2 µl of the CLIP-Vista stock solution to 18 µl of cell lysate. Mix well by pipetting up and down several times.
  5. Incubate in the dark for 60 minutes at 37°C.
  6. 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.
  7. 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.
  8. After fluorescent imaging, standard fixing and staining protocols can be used to detect the non-fluorescent proteins.