https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=Protein-Lipid_Overlay_Assay 2026-06-02T00:37:46Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=Protein-Lipid_Overlay_Assay&diff=560&oldid=prev 2011-01-31T20:55:18Z

<p>added categories</p> <table style="background-color: #fff; color: #202122;" data-mw="interface"> <col class="diff-marker" /> <col class="diff-content" /> <col class="diff-marker" /> <col class="diff-content" /> <tr class="diff-title" lang="en"> <td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td> <td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 20:55, 31 January 2011</td> </tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l10">Line 10:</td> <td colspan="2" class="diff-lineno">Line 10:</td></tr> <tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># Wash the blots and incubate for 1hr at room temperature with a secondary antibody in 5% non-fat dry milk in TBS with 0.1% Tween20.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># Wash the blots and incubate for 1hr at room temperature with a secondary antibody in 5% non-fat dry milk in TBS with 0.1% Tween20.</div></td></tr> <tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># Wash as in (5) &amp; visualize the blot using Super-Signal chemiluminescent kit (Pierce).</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div># Wash as in (5) &amp; visualize the blot using Super-Signal chemiluminescent kit (Pierce).</div></td></tr> <tr><td colspan="2" class="diff-side-deleted"></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td colspan="2" class="diff-side-deleted"></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">[[ Category: Protein-Lipid Interactions ]]</ins></div></td></tr> <tr><td colspan="2" class="diff-side-deleted"></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">[[ Category: Western Blot ]]</ins></div></td></tr> <!-- diff cache key bridgeslabproto-mw_:diff:1.41:old-559:rev-560:php=table --> </table>

Davebrid https://bridgeslab.sph.umich.edu/protocols/index.php?title=Protein-Lipid_Overlay_Assay&diff=559&oldid=prev 2011-01-31T20:54:53Z

<p>copied protocol</p> <p><b>New page</b></p><div>from Emr Lab via Lois Weisman<br /> <br /> <br /> # Prepare serial two-fold dilutions of PIP starting from 0.2mM dissolved in a resuspension solution of CHCl3:CH3OH:50mM HCl (5:10:4, v/v/v) with 2ml Ponceau S (Fluka 09276).<br /> # Cut the proper size of nitrocellulose membrane (GE Water &amp; Process Technologies, WP4HY00010).<br /> # Spot 1ml of the PIP solution on the membrane.<br /> # After drying blots in dark at room temperature (1 hr) &amp; at 4oC (overnight), incubate the blot for 1hr at room temperature in a blocking solution (5% non-fat dry milk in TBS with 0.1% Tween20).<br /> # Then incubate the blot overnight at 4oC with 5mg/ml the bacterial expressed and purified GST-fusion proteins in 0.5% fatty-acid free BSA (Sigma) in TBS with 0.1% Tween20.<br /> # After washing 5 times each 5 minutes with TBS with 0.1% Tween20, incubate the blot overnight in a cold room with anti-GST antibody in a solution of 0.5% fatty-acid free BSA (Sigma) in TBS with 0.1% Tween20.<br /> # Wash the blots and incubate for 1hr at room temperature with a secondary antibody in 5% non-fat dry milk in TBS with 0.1% Tween20.<br /> # Wash as in (5) &amp; visualize the blot using Super-Signal chemiluminescent kit (Pierce).</div>

Davebrid