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Finkelstein Lab Wiki

Filter Binding Assay Protocol

Contents

Section titled “Contents”
  1. Procedure

1. Procedure

Section titled “1. Procedure”
  1. Make Equilibriation Buffer (20 mM TrisHcl pH8, 6 mM NaCl, 5 mM 2-ME-don’t add yet, 1 mM EDTA, 10% glycerol) and make sure to cool to 4C.
  2. Wash BioRad TransBlot Transfer Medium Pure Nitrocellulose Membrane (0.45 um) in 0.4M KOH.
  3. Rinse nitrocellulose in millipore ddH2O twice to normalize pH.
  4. Add 35 ul B-ME to 100 mL Eq Buffer and equilibriate nitrocellulose for at least 1 hr at 4C.
  5. While that incubates, start reactions.
  6. Place 3x SF filter paper, then nitrocellulose into Biodot apparatus and make a small cut at the bottom right hand corner to remember orientation.
  7. Vacuum dry nitrocellulose. Then add 100 ul eq buffer to each well, making sure not to add bubbles. Vacuum all liquid through.
  8. Add each reaction to its subsequent well and vacuum until wells look dry.
  9. Wash all wells with 100 ul Eq buffer again and vacuum dry. Wrap nitrocellulose in plastic wrap and place in phosphoroimager overnight.