Editing PKA alpha purification Feb 2012 (section)

== pSpeedET-PKAalpha-pGKJE8, Top10, 0.02% arabinose, 37 oC,&nbsp; 4 hours, Qiagen column<u><br></u>  ==

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

<u>Culture conditions:</u> <br> 

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

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

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

The following was done on: ____ 

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

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

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

<u>Purification Conditions:</u> pump to Qiagen 5 ml cartridge column, step gradient&nbsp; with peristaltic pump. 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, change buffer then dialyse o/n. <br> 

'''Protein'''<br> 

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

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

<u>Total vol</u>:<br> 

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

<u>Concentration</u>:<br> 

<u>Size:</u> 

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

<u>Purity</u>: Not good, as shown below, probably washing vol is not sufficient.<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>