Bead Preparation
- Take 30-60 µL Neutravidin beads (Peirce), 50% slurry per treatment condition (i.e., compound and control).
- Wash 2-3x in 500 µL lysis buffer.
- 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.
- Add 5 mM biotin, bind with rotation 30 min, 4°C.
- Wash 3x in 500 µL lysis buffer.
- Resuspend in lysis buffer to a total volume of 40 µL per treatment condition.
Cell Lysis and Fractionation
- 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).
- Resuspend in 1-20 mL cold lysis buffer.
- Pipette up and down to lyse cells.
- Spin 14,000xg, 10 minutes.
- Measure concentration of supernatant (Bradford assay).
- Save aliquot for “input” check on gel.
Pulldown
- Add prepared beads to clarified lysate, rotate 1.5 h, 4°C.
- Wash 3x 2 min with lysis buffer, transferring to fresh tube during first wash.
- Remove all liquid with Hamilton syringe.
- Add 20-30 µL 2x LDS sample buffer (Invitrogen) + 5% β-mercaptoethanol.
- Boil 10 minutes.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