Bitan:Biotinylation of antibodies)

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</head>

<body lang=EN-US style='tab-interval:.5in'>

<div class=Section1>

<p class=MsoNormal><span class=SpellE><b style='mso-bidi-font-weight:normal'>Biotinylation</b></span><b style='mso-bidi-font-weight:normal'> of mAb211 <o:p></o:p></b></p>

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>mAb211 stock = [200ug/<span class=SpellE>mL</span>) in PBS</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span></span></span><![endif]>Biotin Labeling Reagent Used: NHS-PEG<span style='font-size:8.0pt;line-height:115%'>12</span>-Biotin (from <span class=SpellE>ThermoScientific</span>)</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Amount of Biotin Reagent Used for 40mmol excess BR (labeled as 2X):</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>0.030mL x 0.2mg/<span class=SpellE>mL</span> x 1mmol <span class=SpellE>IgG</span>/150,000 mg <span class=SpellE>IgG</span> x 40mmol BR/<span class=SpellE>mmol</span> mAb211 = 0.0000016mmol BR</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>0.0000016mmol BR x 1,000,000uL/L x L/250mmol = 0.0064uL BR from original stock\</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Amount of Biotin Reagent Used for 60mmol excess BR (labeled as 3X):</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>0.030mL x 0.2mg/<span class=SpellE>mL</span> x 1mmol <span class=SpellE>IgG</span>/150,000 mg <span class=SpellE>IgG</span> x 60mmol BR/<span class=SpellE>mmol</span> mAb211 = 0.0000024mmol BR</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>0.0000024mmol BR x 1,000,000uL/L x L/250mmol = 0.0096uL BR from original stock</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Take 1uL from original stock vial (use syringe from Aida’s draw under column) </p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Add 999uL DMSO to make 1000X dilution</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;

</span></span></span><![endif]>Always use dry solvent for BR since NHS-ester reactive group is susceptible to hydrolysis with exposure to moisture.</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>6.4 <span class=SpellE>uL</span> of 1000X diluted BR added to 30uL of mAb211 (2X)</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>9.6uL of 1000X diluted BR added to 30uL of mAb211 (3X)</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span></span></span><![endif]>Both <span class=SpellE>eppendorfs</span> were incubated with BR on ice for 2 hours</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span class=SpellE>Unreacted</span> biotin removed by running solution through micro-column</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>To prepare micro-column remove bottom seal and loosen cap</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Centrifuge for 1.5 min at 14000 rpm</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Solution discarded and slanted portion marked</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Sample loaded on column </p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level3 lfo2'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'><span style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp;

</span></span></span><![endif]>2X – approx 36uL</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level3 lfo2'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'><span style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]>3X – approx 40uL</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Sample allowed to absorb on column</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>15uL of PBS used as stacking buffer to fully elute protein since &lt;70uL sample loaded on column.</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Final volumes:</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>2X – approx 52uL (30 + 6.4BR + 15PBS)</p>

<p class=MsoListParagraphCxSpLast style='margin-left:1.0in;mso-add-space:auto; text-indent:-.25in;mso-list:l0 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>3X – approx 55uL (30 + 9.6BR + 15PBS)</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><span class=SpellE><b style='mso-bidi-font-weight:normal'>Biotinylation</b></span><b style='mso-bidi-font-weight:normal'> Check <o:p></o:p></b></p>

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Prepare membrane (Nitrocellulose membrane)</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Do not touch membrane</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;

</span></span></span><![endif]>Cut to appropriate size</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Mark container</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Prepare dilutions of protein sample</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>For 2x and 3X prepare a 10X and 100X dilution</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo4'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'><span style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]>10X: 1uL of original diluted in 9 <span class=SpellE>uL</span> PBS</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo4'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'><span style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]>100X: 1uL of 10X diluted in 9uL PBS</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Prepare blocking reagent. Use advance blocking reagent (1gram in 50mL <span class=SpellE>milliQ</span>) </p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Store at 4 deg C till use</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Blot Protein on membrane (2uL)</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Triplicates for undiluted, 10X dilution, and 100X dilution</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:.75in;mso-add-space: auto'><o:p>&nbsp;</o:p></p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Membrane dried to prevent spread of protein across membrane. </p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Best to keep covered in hood to dry. No need to refrigerate. Prevent accumulation of moisture</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Block with Advance Blocking Reagent ( 2% - 1g in 50mL) for 90 min</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;

</span></span></span><![endif]>Incubate at room temperature on rotator</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Make sure membrane is moving </p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Used 10mL</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Add 1uL of <span class=SpellE>streptavidin</span>

HRP to blocking solution and incubate for 60-75min</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>1:10000 dilution for <span class=SpellE>streptavidin</span> HRP (1uL in 10mL)</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Dilute in blocking buffer</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;

</span></span></span><![endif]>Wash thoroughly: complete 4, 5 min washes </p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Wash 4 times with 10mL PBST for 5 minutes each</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo4'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'><span style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]>PBST: 50mL PBS + 25uL Tween20</p>

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Prepare ECL substrate (reacts with <span class=SpellE>streptavidin</span>-HRP/biotin complex to produce <span class=SpellE>chemiluminence</span> which is then detected by the imager.)</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Take kit out of fridge for at least 10 min</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp; </span></span></span><![endif]>Mix 200uL of each substrate and incubate 5 min with membrane</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo4'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'><span style='mso-list:Ignore'>§<span style='font:7.0pt "Times New Roman"'>&nbsp; </span></span></span><![endif]>Roll solution to spread over entire membrane</p>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo4'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;

</span></span></span><![endif]>Keep ECL substrate wrapped in foil and cover the membrane containing box with foil as well. ELC substrate is highly sensitive to light and will degrade quickly if exposed to light</p>

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo4'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>Image in the Spencer Lab imager</p>

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