https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=Glycogen_Phosphorylase_Assay 2026-06-01T21:11:57Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=Glycogen_Phosphorylase_Assay&diff=589&oldid=prev 2011-09-19T16:44:43Z

<p>typo in 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 16:44, 19 September 2011</td> </tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l27">Line 27:</td> <td colspan="2" class="diff-lineno">Line 27:</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;"><br></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;"><br></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>[[Category: Glycogen]]</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>[[Category: Glycogen]]</div></td></tr> <tr><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: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[[<del style="font-weight: bold; text-decoration: none;">Cagegory</del>: Assays]]</div></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</ins>: Assays]]</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>[[Category: Cell Based Assays]]</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>[[Category: Cell Based Assays]]</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>[[Category: Radioactive Assays]]</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>[[Category: Radioactive Assays]]</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>[[Category: Metabolism]]</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>[[Category: Metabolism]]</div></td></tr> <!-- diff cache key bridgeslabproto-mw_:diff:1.41:old-588:rev-589:php=table --> </table>

Davebrid https://bridgeslab.sph.umich.edu/protocols/index.php?title=Glycogen_Phosphorylase_Assay&diff=588&oldid=prev 2011-09-19T16:42:46Z

<p>copied protocol from alan cheng</p> <p><b>New page</b></p><div>==Materials==<br /> * Homogenization Buffer: 50 mM MES (pH 6.1), 50 mM KF, 60 mM b-mercaptoethanol, 10 mM EDTA, 0.5% Triton X-100 with protease inhibitors<br /> * Ice cold PBS<br /> * Reaction Mixture: 1 mL of 400 mM KF, 10 mM EDTA, 30 mg of glycogen. Warmed at 42C until glycogen is dissolved. <br /> * 200 mM Glucose-1-Phosphate<br /> * 14C Glucose-1-Phosphate<br /> * 100 mM 5&#039;-AMP<br /> * GF/A filter circles<br /> * Ice cold 70% ethanol<br /> <br /> ==Protocol==<br /> # Cells are starved and treated as desired.<br /> # Wash 3x with ice cold PBS.<br /> # 200 uL of homogenization buffer is added per well of a 6 well dish.<br /> # Cells are scraped then centrifuged at 13k for 10min. Lysates are transfered to fresh tubes.<br /> # Prepare radioactive reaction mixture. <br /> ## Need 2 x 40 uL reaction mixture per sample.<br /> ## Add 25 uL of 200 mM G1P and 2 uCi of radioactive G1P to 1mL Reaction Mixture. <br /> ## Assays are done +/- 3 mM 5&#039;-AMP. Prepare one reaction mixture tube with and with 45 uL/mL AMP and one with water.<br /> # Add 20 uL Lysate to 40 uL radioactive reaction mixture.<br /> # Place at 37C for 20 min to react.<br /> # Place samples on ice for 15 min.<br /> # Spot 50 uL of mixture onto GF/A filter and let air dry for a few seconds. Place in a 6 well dish.<br /> # Add ice cold 70% Ethanol and washed 15-20 min at 4C.<br /> # Wash twice more with room temperature 70% ethanol for 15-20 min.<br /> # Air dry and count filters.<br /> <br /> [[Category: Glycogen]]<br /> [[Cagegory: Assays]]<br /> [[Category: Cell Based Assays]]<br /> [[Category: Radioactive Assays]]<br /> [[Category: Metabolism]]</div>

Davebrid