Bitan:Biotinylation of antibodies)



    

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

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

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

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

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

<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 <![endif]>0.030mL x 0.2mg/ mL x 1mmol IgG /150,000 mg IgG x 40mmol BR/ mmol mAb211 = 0.0000016mmol BR

<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 <![endif]>0.0000016mmol BR x 1,000,000uL/L x L/250mmol = 0.0064uL BR from original stock\

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

<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 <![endif]>0.030mL x 0.2mg/ mL x 1mmol IgG /150,000 mg IgG x 60mmol BR/ mmol mAb211 = 0.0000024mmol BR

<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 <![endif]>0.0000024mmol BR x 1,000,000uL/L x L/250mmol = 0.0096uL BR from original stock

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

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

<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

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

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

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

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

<![endif]>Both eppendorfs were incubated with BR on ice for 2 hours

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

<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 <![endif]>To prepare micro-column remove bottom seal and loosen cap

<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 <![endif]>Centrifuge for 1.5 min at 14000 rpm

<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 <![endif]>Solution discarded and slanted portion marked

<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 <![endif]>Sample loaded on column

<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'> §

<![endif]>2X – approx 36uL

<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'> § <![endif]>3X – approx 40uL

<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 <![endif]>Sample allowed to absorb on column

<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 <![endif]>15uL of PBS used as stacking buffer to fully elute protein since &lt;70uL sample loaded on column.

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

<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 <![endif]>2X – approx 52uL (30 + 6.4BR + 15PBS)

<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 <![endif]>3X – approx 55uL (30 + 9.6BR + 15PBS)

<p class=MsoNormal><o:p> </o:p>

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

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

<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 <![endif]>Do not touch membrane

<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

<![endif]>Cut to appropriate size

<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 <![endif]>Mark container

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

<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 <![endif]>For 2x and 3X prepare a 10X and 100X dilution

<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'> § <![endif]>10X: 1uL of original diluted in 9 <span class=SpellE>uL PBS

<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'> § <![endif]>100X: 1uL of 10X diluted in 9uL PBS

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

<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 <![endif]>Store at 4 deg C till use

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

<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 <![endif]>Triplicates for undiluted, 10X dilution, and 100X dilution

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

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

<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 <![endif]>Best to keep covered in hood to dry. No need to refrigerate. Prevent accumulation of moisture

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

<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

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

<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 <![endif]>Make sure membrane is moving

<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 <![endif]>Used 10mL

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

HRP to blocking solution and incubate for 60-75min

<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 <![endif]>1:10000 dilution for streptavidin HRP (1uL in 10mL)

<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 <![endif]>Dilute in blocking buffer

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

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

<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 <![endif]>Wash 4 times with 10mL PBST for 5 minutes each

<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'> § <![endif]>PBST: 50mL PBS + 25uL Tween20

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

<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 <![endif]>Take kit out of fridge for at least 10 min

<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 <![endif]>Mix 200uL of each substrate and incubate 5 min with membrane

<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'> § <![endif]>Roll solution to spread over entire membrane

<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

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