Immunoprecipitation of ROR1

Materials and Reagents

1. ROR1-positive cells (e.g. RCH-ACV cells, Kasumi-2 cells, 697 cells)
   
2. Cell lysis buffer (Cell Signaling Technology, catalog number: 9803 )
   
3. Protease inhibitor cocktail (cOmplete Mini EDTA-free) (Roche, catalog number: 04693159001 )
   
4. Phosphatase inhibitor cocktail II (Sigma-Aldrich, catalog number: P5726 )
   
5. Antibody specific for ROR1 (R&D Systems, catalog number: AF2000 )
   
6. Isotype matched control (R&D Systems, catalog number: AB-108-C )
   
7. Protein G Agarose beads (EMD Millipore, catalog number: 16-266 )
   
8. SDS
   
9. BSA
   
10. DTT
    

Equipment

1. P1000 pipette
   
2. Centrifuge
   

Procedure

1. 10⁷ cells (such as RCH-ACV, Kasumi-2, 697 cells) are pelleted, washed 1x in PBS, pelleted, and thoroughly resuspended in 500 μl ice-cold lyses buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail II (according to manufacturers’ instruction) using a P1000 pipette.
   
2. The lysis reaction is kept on ice and vortexed at max speed 3 times for 3-second bursts. The reaction is kept on ice for 5 min between each vortexing.
   
3. Following lysis, samples are centrifuged at max-speed for 15 min at 4 °C.
   
4. Following pelleting, the supernatant is transferred to a fresh tube, kept on ice, and subjected to Bradford protein concentration analysis. Lysate is diluted to 2 mg/ml in lysis buffer for IP.
   
5. 3 μg of antibody specific for ROR1 or an isotype matched control was incubated with cell extracts (500 μl at 2 mg/ml) by rotating overnight.
   
6. Protein G Agarose beads were prepared for precipitation by washing twice in lysis buffer and blocking for 1 h (nutating) at 4 °C in lysis buffer containing 0.5% BSA.
   
7. Following blocking, beads were pelleted and resuspended in lysis buffer to a 50% slurry, and 40 μl 50% slurry was added to lysate and rocked at 4 °C for 1 h.
   
8. Following precipitation, beads were washed in 500 μl lysis buffer 3x by nutating at 4 °C for 20 min.
   
9. Proteins were eluted from the beads by boiling in an aqueous solution with 2% SDS and 25 mM DTT.
   

Acknowledgments

This protocol was adapted from Bicocca et al. (2012).

References

1. Bicocca, V. T., Chang, B. H., Masouleh, B. K., Muschen, M., Loriaux, M. M., Druker, B. J. and Tyner, J. W. (2012). Crosstalk between ROR1 and the Pre-B cell receptor promotes survival of t(1;19) acute lymphoblastic leukemia. Cancer Cell 22(5): 656-667.