HTNCre Purification Feb 2012

pET28b-HTNCre, NiCo21, 37 oC,  4 hours

Clone: pET28b-HTNCre, clone # 298

Cells: NiCo21 transformed with  pET28b-Cre

Culture conditions:

Innoculate 1 L LB/K with a single colony of pET28b-HTNCre NiCo21. Grow 8 hours at 37 oC.

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

Induction: at OD about 0.8, add 1 ml of 1 M IPTG to 1 L culture, induce at 37 oC for 4 hours

Cell pellet wet weight:  Some cells appear to be dying after induction,  total 27.4 g

Lysis: Freeze then thaw one cycle

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

Purification Conditions: 10 ml Ni column, 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

Lot number on Label:

Total vol:

Total amount of protein mg:

Concentration:

Size:

Total number of tubes:

Purity:

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.

Location:

pET28b-HTNCre, NiCo21, 37 oC, 2 hours.

Cells: NiCo21 transformed with pET28b-HTNCre.

Culture conditions:

Innoculate 1 L LB/K/Chl with a single colony of NiCo21 cells from plate. Grow 8 hours.

Next day add 100 ml culture to 1 L LB/K, 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)

Lysis: Freeze and thaw one time

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 "HTNCre--Paul, 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.

pET28b-HTNCre, NiCo21, 37 oC,  1.5 hours

Clone: pET28b-HTNCre, clone # 298

Cells:  NiCo21 transformed with  pET28b-Cre

Culture conditions:

Innoculate 1 L LB/K/Chl with a single colony of pET28b-HTNCre NiCo21. Grow o/n at 37 oC.

Next day add 100 ml to 1 L LB/K/Chl, grow at 37 oC. Grow 7 L

Induction: at OD about 0.8, add 0.5 ml of 1 M IPTG to 1 L culture, induce at 37 oC for 1.5 hours

Cell pellet wet weight:  cells appear to be healthy,  total 22.4 g (2/14/2012)

Lysis: Freeze, thaw.

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

Purification Conditions:

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.

Buffers

Lysis Buffer:

Wash Buffer:

Elution Buffer:

Dialysis Buffer: