https://eln.excellgen.com/index.php?action=history&feed=atom&title=PKA_alpha_purification_Feb09_2012PKA alpha purification Feb09 2012 - Revision history2026-10-18T09:29:56ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=PKA_alpha_purification_Feb09_2012&diff=6658&oldid=prevPaul.xu at 17:25, 22 October 20122012-10-22T17:25:33Z<p></p>
<p><b>New page</b></p><div>== pSpeedET-PKAalpha, NiCo21, 0.02% arabinose, 37 oC, 3 hours<br> ==<br />
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Cells: NiCo21 transformed with pSpeedET-PKAalpha. Grow on LB/K plate o/n <br />
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Culture conditions: <br />
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Innoculate 1 L LB/K with a single colony. Grow o/n.---02-09-2012 <br />
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Next day add 100 ml culture to 1 L LB/K, grow at 37 oC.---02-10-2012 <br />
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Grow cells in 1 L LB/Kl to about OD600 = 0.8 at 37 oC.<br> <br />
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Induction: induce at RT with 2 ml of 10% arabinose to 1 L culture and induce 4 hours: ---9:20am; 02-10-2012<br> <br />
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Pellet: white with very little dead cells, total wet weight from 3 L: '''35 g''' <br />
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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 <br />
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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. <br />
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Purification Conditions: <br />
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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). <br />
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Manual step gradient, wash (20 mM imidazole) until no detectable protein, elute (300 mM imidazole), collect 3 ml fractions <br />
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Dialyse: 1 X TBS, 50% glycerol, 6 hours, change buffer then dialyse o/n. <br />
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Protein <br />
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Buffer: 1 X TBS, 50% glycerol <br />
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Name on Label: <br />
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Lot number on Label: <br />
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Storage: 80 oC freezer (storage room) in a freezer box labeled as "Purified PKAalpha Protein, 2/2012" <br />
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Total vol: <br />
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Total amount of protein mg: <br />
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Concentration: <br />
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Size: <br />
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Total number of tubes: <br />
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Purity: <br />
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<br>Samples: 15 ul from 300 mM imidazole elution fractions (combined) <br />
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MW ladder: <br />
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ProteinOne <br />
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Gel: Invitrogen Bis-Tris NuPAGE 4-12%, 10 wells <br />
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Buffer: <br />
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Stain: ProteinOne <br />
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Destain: water.<br> <br />
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=== Buffers ===<br />
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Lysis Buffer: <br />
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Wash Buffer: <br />
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Elution Buffer: <br />
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Dialysis Buffer:<br> <br />
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[[Category:Protein_Purification]]</div>Paul.xu