PKA alpha purification Feb09 2012

pSpeedET-PKAalpha, NiCo21, 0.02% arabinose, 37 oC, 3 hours

Cells: NiCo21 transformed with pSpeedET-PKAalpha. Grow on LB/K plate o/n

Culture conditions:

Innoculate 1 L LB/K with a single colony. Grow o/n.---02-09-2012

Next day add 100 ml culture to 1 L LB/K, grow at 37 oC.---02-10-2012

Grow cells in 1 L LB/Kl to about OD600 = 0.8 at 37 oC.

Induction: induce at RT with 2 ml of 10% arabinose to 1 L culture and induce 4 hours: ---9:20am; 02-10-2012

Pellet: white with very little dead cells, total wet weight from 3 L: 35 g

Lysis: Freeze and thaw one time, cells were completely lysed after one freeze-thawing cycle. Use homogenizer to break sticky genomic DNA.---02--12-2012

Preclear: Centrifuge 12000 rpm for 15 min, resuspend pellet in lysis buffer, homogenize, and spin again at 12000 rpm for 15 min. Combine supernatant and spin at 25000 rpm for 20 min.

Purification Conditions:

add 10 ml Ni beads to clarified supernatant, incubate in a beaker with stir bar for 1 hour in cold room, spin down beads at 1000 rpm for 1 min, collect supernatant (do not discard), transfer beads to a glass column using washing buffer (20 mM imidazole).

Manual step gradient, wash (20 mM imidazole) until no detectable protein, elute (300 mM imidazole), collect 3 ml fractions

Dialyse: 1 X TBS, 50% glycerol, 6 hours, change buffer then dialyse o/n.

Protein

Buffer: 1 X TBS, 50% glycerol

Name on Label:

Lot number on Label:

Storage: 80 oC freezer (storage room) in a freezer box labeled as "Purified PKAalpha Protein, 2/2012"

Total vol:

Total amount of protein mg:

Concentration:

Size:

Total number of tubes:

Purity:

Samples: 15 ul from 300 mM imidazole elution fractions (combined)

MW ladder:

ProteinOne

Gel: Invitrogen Bis-Tris NuPAGE 4-12%, 10 wells

Buffer:

Stain: ProteinOne

Destain: water.

Buffers

Lysis Buffer:

Wash Buffer:

Elution Buffer:

Dialysis Buffer: