Paul.xu at 18:14, 11 June 2011

2011-06-11T18:14:35Z

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1. Centrifuge lysate for 30 min at 12000 RPM. Carefully transfer supernatant to a new tube (no particles or turbid stuff). 

2. If you accidentally "contaminate" supernatant with some loose "pellet", filter the supernatant with 0.45 u filter. 

3. Load the supernatant onto an 5 ml Ni-NTA column (pre-equilibrated with wash buffer) using a peristolic pump at a speed 0.5 ~1 ml/min. 

4. Wash column with 30 ml filtered wash buffer containing 20 ~50 mM imidazole. 

5. Connect the column to AKTA FPLC. Use program #_ to wash and elute proteins. Collect 2 ml/minute fractions.

6. Analyze fractions from the initial flowthrough (wash) as well as fractions from the elution that contain protein.

7. For each fraction, dilute 15 ul of sample with  3 ul of 6 X SDS sample buffer. Boil at 95 oC for 2 min and load  onto a 10% SDS-PAGE gel. Run at 200 V for 35 min. Stain the gel with coomassie.

8. Estimate the amount of protein in each fraction using Bradford reagent. Pool the fractions that contain PKA alpha. Adjust protein concentration to 2.5 mg/ml using low concentration fractions. Dialyze PKA alpha with 2X storage buffer. 

9. Measure protein concentration using Bradford reagent and OD280.

10. Add DTT (final concentration 2 mM) and glycerol (final concentration 50%) ( optional: 50 mM trehalose). 

11. Dispense PKA into 1 ml (2 mg/ml) aliquots, quick freeze in a dry ice/ethanol bath and store at â80oC.

12. Test the activity of the enzyme using Kemptide (PKA Peptide Substrate, LRRASLG(Leu-Arg-Arg-Ala-Ser-Leu-Gly) and luciferase. 

[[Category:Protocols]]

PKA FPLC-Purification - Revision history

Paul.xu at 18:14, 11 June 2011