Metal Chelate Affinity Chromatography

Connect chelating column to pump C and wash out preservatives with 2 column volumes of water

Charge with 5 column volumes of 200mM CoCl₂ at 1ml/min

Wash with water until eluent is clear and column is uniform pink

Equilibrate with buffer A without DTT and imidazole

Load sample at 0.5 ml/min and collect flow through (may repeat loading)

Wash sample holder with a little buffer and load last bit over column

Wash pumps A and B with appropriate buffers and program gradient

Connect pump A and B lines to column and begin washing and elution (#2)

Method

0.00 CONC % B 0.0

0.00 ML/MIN 0.50

0.00 CM/ML 0.50

0.00 PORT.SET 6.1

0.00 VALVE.POS 1.1

15.00CONC %B 0.0

15.00 ML/MIN 0.50

15.00 CM/ML 0.50

15.00 PORT.SET 6.1

15.00 VALVE.POS 1.1

60.00 CONC %B 100

60.00 ML/MIN 0.50

60.00 CM/ML 0.50

60.00 PORT.SET 6.1

60.00 VALVE.POS 1.1

75.00 CONC %B 100

75.00 ML/MIN 0.50

75.00 CM/ML 0.50

75.00 PORT.SET 6.0

75.00 VALVE.POS 1.1

76.00 CONC %B 0.0

76.00 ML/MIN 0.50

76.00 CM/ML 0.50

76.00 PORT.SET 6.0

76.00 VALVE.POS 1.1

90.00 CONC %B 0.0

90.00 ML/MIN 0.50

90.00 CM/ML 0.50

90.00 PORT.SET 6.0

90.00 VALVE.POS 1.1

Solutions:

Phosphate Buffer A

50mM Na-PO₄ pH 8.0 46.6ml 1M Na₂ PO₄ + 3.4ml 1M NaH₂PO₄

300mM NaCl 60ml 5M

10% glycerol 100ml

Phosphate Buffer B

50mM Na-PO₄ pH 8.0 46.6ml 1M Na₂ PO₄ + 3.4ml 1M NaH₂PO₄

300mM NaCl 60ml 5M

10% glycerol 100ml

400mM imidazole 27.23g

Lysis Buffer 500ml

20mM Na-PO₄ 8.96ml Na₂PO₄ + 1.04ml NaH₂PO₄ pH 7.8

20mM NaCl 2ml 5M

1mM PMSF

1mg/ml leupeptin

2mg/ml aprotinin pH 7.8

1M Na-PO₄ Bufferà932ml 1M Na₂PO₄ + 68ml 1M NaH₂PO₄

Lyse and dounce cells in lysis buffer 1ml/150mm plate

Bring Na-PO₄ to 50mM and NaCl to 300mM

Add 560ml per 10ml(5M NaCl) Add 300ml 1M pH 7.8 Na-PO₄

Dounce cells and spin at 45K for 1h

Load over the column

Collect fractions containing the desired protein from chelating column

Dilute 6 fold with Buffer C (0M NaCl) + 50mM NaCl

Apply to 1ml Hitrap Q equilibrated in C + 50 mM NaCl

Wash with 10ml C + 50 mM NaCl

Elute with linear gradient from 50-500mM NaCl in C

Cyclin H-His elutes ~300mM

Buffer C + 50 mM NaCl 1liter

25mM Hepes pH 7.4 25ml 1M

50mM EDTA 2ml 0.5M

50mM NaCl 10ml

10% glycerol 100ml

Add 1mM DTT

Buffer C + 500 mM NaCl 1liter

25mM Hepes pH 7.4 25ml 1M

50mM EDTA 2ml 0.5M

50mM NaCl 100ml

10% glycerol 100ml

Add 1mM DTT