PKA alpha purification Feb 2012

pSpeedET-PKAalpha-pGKJE8, Top10, 0.02% arabinose, 37 oC,  4 hours, Qiagen column

Cells: Top10 transformed with pSpeedET-PKAalpha and pGKJE8 (2011). Recover single colony on LB/K/Chl plate by streaking from glycerol stock.

Culture conditions:

Innoculate 1 L LB/K/Chl with a single colony of Top10 cells from plate (streak from glycerol stock). Grow o/n.

Next day add 100 ml to 1 L LB/K/Chl, grow at 37 oC.

Induction: at OD about 0.8, add 2 ml of 10% arabinose to 1 L culture, induce at 37 oC for 4 hours

The following was done on: ____

Cell pellet wet weight: Cells grow fine with LB/K/Chl,  ~ 1.5 g per L, pellet is white (very little dead cells).

Lysis: Freeze, thaw, add lysis 50 ml buffer and 30 ul lysozyme ,  digest at RT for 30 min, then add 10 ul DNase,  digest put on rotary mixer o/n.

Preclear: Centrifuge 12000 rpm for 15 min, then 25000 rpm for 20 min.

Purification Conditions: pump to Qiagen 5 ml cartridge column, step gradient  with peristaltic pump. wash (20 mM imidazole) , elute (300 mM imidazole), collect 3 ml fractions

Dialysis: 1 X TBS, 50% glycerol, 70 ml, 6 hours, change buffer then dialyse o/n.

Protein

Buffer: 1 X TBS, 50% glycerol

Lot number on Label:

Total vol:

Total amount of protein mg:

Concentration:

Size:

Total number of tubes:

Purity: Not good, as shown below, probably washing vol is not sufficient.

Samples: 15 ul from  300 mM  imidazole elution fractions (3 ml each)

MW ladder: 250, 130, 100, 70(red), 55, 35, 25(red), 15, 10(green)

FERMENTAS PageRuler™ Plus Prestained Protein Ladder

Gel: Invitrogen NuPAGE Bis-Tris NuPAGE 4-12%, 1.0 mm X 10 wells

Buffer:  1X Mes-Tris-SDS. (50 mM Mes, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH7.3), diluted from 20 X Mes-SDS-Running buffer from Boston BioProducts

Stain: Invitrogen Simple Blue

Destain: water.

pSpeedET-PKAalpha, Top10, 0.02% arabinose, RT (25 ~30 oC) o/n.

Cells:Top10 transformed with pSpeedET-PKAalpha and chaperone plasmids (2011). Recover single colony on LB/K/Chl plate by streaking from glycerol stock.

Culture conditions:

Innoculate 1 L LB/K/Chl with a single colony of Top10 cells from plate (streak from glycerol stock). Grow o/n.

Next day add 100 ml culture to 1 L LB/K/Chl, grow at 37 oC.

Grow cells in 1 L LB/K/Chl to about OD₆₀₀ = 0.8 at 37 oC, then cool down to RT.

Induction: induce at RT with 1 ml of 10% arabinose to 1 L culture and induce overnight

Pellet: some dead cells (black)

Name	PGn7	PGKJE8	PKJE7	PTf16	PG-Tf2
Weight (g)	2.6	1.3	0.9	1.1	3.7

combined pellets

Lysis: Freeze and thaw one time, add lysis 50 ml buffer and 30 ul lysozyme (lysozyme did not seem to produce sticky DNA. Looks like most cells are dead), use sonicator to disrupt cells.

Preclear: Centrifuge 12000 rpm for 15 min, then 25000 rpm for 20 min.

Purification Conditions:

add 10 ml Ni beads to clarified supernatant, incubate (rotary) for 1 hour in cold room, spin down beads at 1000 rpm for 1 min, remove supernatant, transfer beads to glass column.

Manual step gradient, wash (20 mM imidazole) , elute (300 mM imidazole), collect 3 ml fractions

Dialysis: 1 X TBS, 50% glycerol, 70 ml, 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.

pSpeedET-PKAalpha, NiCo21, 0.02% arabinose, 37 oC, 4 hours.

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

Culture conditions:

Innoculate 1 L LB/K/Chl with a single colony. Grow o/n.

Next day add 100 ml culture to 1 L LB/K/Chl, grow at 37 oC.

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

Induction: induce at RT with 1 ml of 10% arabinose to 1 L culture and induce 4 hours

Pellet: white with very little dead cells, total wet weight from 6 L: 18 g

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

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: