Faculty Research Labs

Pulldown of Compound Targets using Biotinylated Affinity Reagents: Whole-cell Lysate Method

Bead Preparation

  1. Take 30-60 µL Neutravidin beads (Peirce), 50% slurry per treatment condition (i.e., compound and control).
  2. Wash 2-3x in 500 µL lysis buffer.
  3. Add 100 µM affinity reagent or linker control in an appropriate volume of lysis buffer (200-300 µL), bind with rotation 30 min, 4°C.
  4. Add 5 mM biotin, bind with rotation 30 min, 4°C.
  5. Wash 3x in 500 µL lysis buffer.
  6. Resuspend in lysis buffer to a total volume of 40 µL per treatment condition.

Cell Lysis and Fractionation

  1. Thaw cell pellet on ice (may use 5L equivalent pellet of HeLa S3 cells or 1-2 15 cm plate equivalents of adherent cells such as MCF7).
  2. Resuspend in 1-20 mL cold lysis buffer.
  3. Pipette up and down to lyse cells.
  4. Spin 14,000xg, 10 minutes.
  5. Measure concentration of supernatant (Bradford assay).
  6. Save aliquot for “input” check on gel.

Pulldown

  1. Add prepared beads to clarified lysate, rotate 1.5 h, 4°C.
  2. Wash 3x 2 min with lysis buffer, transferring to fresh tube during first wash.
  3. Remove all liquid with Hamilton syringe.
  4. Add 20-30 µL 2x LDS sample buffer (Invitrogen) + 5% β-mercaptoethanol.
  5. Boil 10 minutes.
  6. Analyze by SDS-PAGE followed by silver staining and/or immunoblot.

Recipe

Lysis Buffer

20 mM Tris, pH 8.0
150 mM NaCl
20 µM leupeptin
1 mM PMSF
1 mM NaF
1 mM β-glycerophosphate
2 mM sodium orthovanadate
2 mM EDTA
10% glycerol
1% NP-40