https://eln.excellgen.com/index.php?action=history&feed=atom&title=1382-1406_Purification1382-1406 Purification - Revision history2026-10-18T09:33:30ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=1382-1406_Purification&diff=6945&oldid=prevPaul.xu at 16:03, 9 November 20122012-11-09T16:03:13Z<p></p>
<p><b>New page</b></p><div>'''1382-1406 purification on 10-30-2012 by Xiuli '''<br />
'''Transfection and cell viability''':<br />
Paul did the transfection directly in the shaking flasks. The cells had very poor viability (shake didn't run properly) when collected.&nbsp; Xiuli did transfections in the plate, cells started recovery on 3 rd day from transfection (didn't change into serum free medium, cells contaminated that week) and&nbsp; stayed in the serum medim until collection.<br> <br />
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Collect 30 ml Serum-free medium from each flasks (5)&nbsp;and and&nbsp; plates (5) 20 ml and centriguged at 12000rpm for 15 mins. <br />
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'''Supernants:''' <br />
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Incubate supernanats (Paul's sample ) with protein A beads, washed with PBS and elute with 0.2 mM Glycine (pH=2.0), elution volume: 900 ul<br> <br />
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'''Pellet:''' <br />
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Lysed pellet with 0.1% TX114 in PBS and collected supernant at 12000 rpm for 20 mins. Incubated the supernatants with protein A beads. Washed with PBS and eluted with Glycine (pH=2.0) for 4 times.<br> <br />
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Load 20-35 ul on the 4-20% SDS gel <br />
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Run Paul's Sup and lysate samples, no protein show on the gel, Run Xiuli's samples from lysate. <br />
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[[Image:1382-1406 10-30-2012.jpg]] <br />
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IgG Pull down seems right, Lane 14 for 1383 have a faint band, can be the right size.<br> <br />
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[[Category:Protein_Purification]]</div>Paul.xu