Editing Taq Purification March 2012

== <span style="text-decoration: underline;">Taq Inactivation modif</span><br>  ==

[[Image:PKAalpha--02-07-2012.jpg|290x266px|PKAalpha--02-07-2012.jpg]] 

Samples: 15 ul from&nbsp; 300 mM&nbsp; imidazole elution fractions (3 ml each)<br> 

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

FERMENTAS PageRulerâ¢ Plus Prestained Protein Ladder<br> 

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

Buffer:&nbsp; 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<br> 

Stain: Invitrogen Simple Blue <br> 

Destain: water.<br>

== pSpeedET-PKAalpha, Top10, 0.02% arabinose, RT (25 ~30 oC) o/n.<u><br></u>  ==

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

<u>Culture conditions</u>: 

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.<br> 

Grow cells in 1 L LB/K/Chl to about OD<sub>600</sub> = 0.8 at 37 oC, then cool down to RT. <br> 

<u>Induction</u>: induce at RT with 1 ml of 10% arabinose to 1 L culture and induce overnight<u><br></u> 

<u>Pellet</u>: some dead cells (black)<br> 

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 455px; height: 80px;"
|-
| Name 
| PGn7 
| PGKJE8 
| PKJE7 
| PTf16 
| PG-Tf2
|-
| Weight (g) 
| 2.6 
| 1.3 
| 0.9 
| 1.1 
| 3.7
|}

combined pellets<br> 

<u>Lysis</u>: 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. 

<u>Preclear:</u> Centrifuge 12000 rpm for 15 min, then 25000 rpm for 20 min. 

<u>Purification Conditions</u>: <br> 

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. <br> 

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

<u>Dialysis:</u> 1 X TBS, 50% glycerol, 70 ml, 6 hours,&nbsp;&nbsp; change buffer then dialyse o/n. <br> 

'''Protein''' 

<u>Buffer</u>: 1 X TBS, 50% glycerol<br> 

<u>Name on Label:</u><br> 

<u>Lot number on Label:</u> 

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

<u>Total vol</u>: 

<u>Total amount of protein mg</u>: 

<u>Concentration</u>: 

<u>Size</u>:<br> 

<u>Total number of tubes:</u><br> 

<u>Purity</u>: <br> 

[[Image:PKAAlapha 2 10 2012.jpg]]<br> 

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

MW ladder: <br> 

ProteinOne 

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

Buffer:&nbsp;&nbsp; 

Stain: ProteinOne 

Destain: water.<br> 

== pSpeedET-PKAalpha, NiCo21, 0.02% arabinose, 37 oC, 4 hours.<br>  ==

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

<u>Culture conditions</u>: 

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. 

<u>Induction</u>: induce at RT with 1 ml of 10% arabinose to 1 L culture and induce 4 hours<br> 

<u>Pellet</u>: white with very little dead cells, total wet weight from 6 L: 18 g<br> 

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

<u>Preclear:</u> 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. 

<u>Purification Conditions:</u> 

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 

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

'''Protein''' 

<u>Buffer</u>: 1 X TBS, 50% glycerol<br> 

<u>Name on Label:</u><br> 

<u>Lot number on Label:</u><br> 

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

<u>Total vol:</u> 

<u>Total amount of protein mg:</u> 

<u>Concentration:</u> 

<u>Size:</u> 

<u>Total number of tubes:</u><br> 

<u>Purity: </u><br> 

[[Image:PKAalpha 2 13 2012.jpg]]<br> 

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

MW ladder: 

ProteinOne 

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

Buffer: <br> 

Stain: ProteinOne 

Destain: water.<br> 

<br> 

== Buffers<br>  ==

Lysis Buffer:<br> 

Wash Buffer:<br> 

Elution Buffer:<br> 

Dialysis Buffer:<br> 

[[Category:Protein_Purification]]