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2.8: Session 8
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- H396P Abl(229-511) Protein Concentration.
Using a syringe, transfer the dialyzed protein solution from the dialysis cassette into a 15- mL conical tube. The cassette cannot be reused and should be discarded after use.
Concentrate the protein solution to a final volume of approximately 500 µL using a Millipore 10-KDa MWCO centrifugal concentrator. For concentration, add up to 12 mL at a time of your dialyzed protein solution to the concentrator tube and replace the cap. Place the concentrator tube with the volume gradations facing up in the centrifuge. Add a counterbalance, either another group’s sample or a 50-mL conical tube with the appropriate volume of water to balance your concentrator tube. Spin at 500 x g until you reach the desired volume, approximately 15 to 45 minutes. Use a pipette to recover your concentrated protein from the filter unit in the concentrator tube. For maximum protein recovery, withdraw the protein from the bottom of the filter unit and use a “side-to-side sweeping motion”. You should remove the concentrated protein solution as soon as possible following centrifugation.
Store your protein in a clearly labeled eppendorf at 4 °C for use in Sessions 13 and 14.
- Protein Quantification
Use the Bio-Rad quantification assay (Session 4) to determine the protein concentration of the H396P Abl kinase domain after purification, dialysis, and concentration. See Appendix A1 for a review on preparing your BSA samples from the 1 mM BSA stock you made in Session 4.
Clearly label the dialyzed protein with your names, your TA’s name, and the date. Store the solution at 4 °C.