https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=Quantification_by_Absorbance_at_280nm 2026-05-31T01:26:36Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=Quantification_by_Absorbance_at_280nm&diff=98&oldid=prev 2009-05-11T14:03:22Z

<p></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 14:03, 11 May 2009</td> </tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l3">Line 3:</td> <td colspan="2" class="diff-lineno">Line 3:</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>#Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer.</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>#Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer.</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>#Calculate extinction coefficient for protein of interest</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>#Calculate extinction coefficient for protein of interest</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>:#Use the [http://www.expasy.ch/tools/protparam.html<del style="font-weight: bold; text-decoration: none;">|</del>Protparam] tool to calculate the extinction coefficient in both molar and mg/mL for protein.  Assume all Cys residues are half-cysteines.</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>:#Use the [http://www.expasy.ch/tools/protparam.html Protparam] tool to calculate the extinction coefficient in both molar and mg/mL for protein.  Assume all Cys residues are half-cysteines.</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>:#If the protein is a GST-fusion add 43110 M&lt;sup&gt;-1&lt;/sup&gt; and 1.607 mg/mL&lt;sup&gt;-1&lt;/sup&gt; respectively</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>:#If the protein is a GST-fusion add 43110 M&lt;sup&gt;-1&lt;/sup&gt; and 1.607 mg/mL&lt;sup&gt;-1&lt;/sup&gt; respectively</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>#Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient</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>#Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient</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>#If necessary adjust by the percent purity as described in [[Determining Percent Purity]]</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>#If necessary adjust by the percent purity as described in [[Determining Percent Purity]]</div></td></tr> <!-- diff cache key bridgeslabproto-mw_:diff:1.41:old-97:rev-98:php=table --> </table>

Davebrid https://bridgeslab.sph.umich.edu/protocols/index.php?title=Quantification_by_Absorbance_at_280nm&diff=97&oldid=prev 2009-05-11T14:02:25Z

<p>wrote initial page</p> <p><b>New page</b></p><div>This method is most accurate with highly purified proteins.<br /> <br /> #Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer.<br /> #Calculate extinction coefficient for protein of interest<br /> :#Use the [http://www.expasy.ch/tools/protparam.html|Protparam] tool to calculate the extinction coefficient in both molar and mg/mL for protein. Assume all Cys residues are half-cysteines.<br /> :#If the protein is a GST-fusion add 43110 M&lt;sup&gt;-1&lt;/sup&gt; and 1.607 mg/mL&lt;sup&gt;-1&lt;/sup&gt; respectively<br /> #Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient<br /> #If necessary adjust by the percent purity as described in [[Determining Percent Purity]]</div>

Davebrid