Daniel:Protocols/AbOligoLink: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Antibody-Oligo Linking= Back to Main This page describes the protocol for antibody-oligo binding used in Combo-lock. The protocol is the first step in ...")
 
>Djacobse
 
(8 intermediate revisions by the same user not shown)
Line 5: Line 5:
This page describes the protocol for antibody-oligo binding used in Combo-lock.  The protocol is the first step in creating antibody probes.  The antibodies are generally polyclonal antibodies from R&D systems.  The protocol uses Sulfo-SMCC, which is a chemical that puts a reactive group on the terminal amine of the antibody.  The oligos used have a thiol group that reacts with the Sulfo-SMCC activated antibody and links the oligonucleotide to the antibody.
This page describes the protocol for antibody-oligo binding used in Combo-lock.  The protocol is the first step in creating antibody probes.  The antibodies are generally polyclonal antibodies from R&D systems.  The protocol uses Sulfo-SMCC, which is a chemical that puts a reactive group on the terminal amine of the antibody.  The oligos used have a thiol group that reacts with the Sulfo-SMCC activated antibody and links the oligonucleotide to the antibody.


==Protocol==
===Buffers===


===Antibody Activation===
<ol>
<li>Prep 100 mL of sodium phosphate buffer</li>
<ol type="A">
<li>Make 100 mL of NaH2PO4 (monobasic) by adding 13.8g to nfH2O</li>
<li>Make 100 mL of Na2H2PO4 (dibasic) by adding 14.2g to nfH2O</li>
<li>Mix 28 mL of monobasic and 72 mL of dibasic to make 1 M (buffer pH 7.2)</li>
<li>Directions from [http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8303.full?text_only=true Cold Spring Harbor]
</ol></ol>


#Resuspend antibodies at 1 mg/mL in PBS.
==Protocol==
#Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3
##250 uL, spin at 1000xg for 2 min
#Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate
#Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer
#Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing
#Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
#Transfer antibodies to new column and spin for 3 min at 1000xg
 
===Oligonucleotide Activation===


#Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA
The protocol is taken (and slightly modified) from the [[Media:Assarsson_Homogenous_PEA_Immunoassay_PLOS_One_2014.PDF|Assarsson PEA Paper]]
#Add 1.3 uL oligonucleotide to tube (or plate)
#Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C
#Add 20 uL PBS with 20 mM EDTA and
#Remove excess DTT  using two consecutive 7 kDa columns equilibrated with 100 mM PBS


===Ab-Oligo Mixing===
Additional paper provides similar but more detailed protocol [[Media:Weibrecht_Visualising_Protein-DNA_Interactions_in_situ_2012_New_Biotech.pdf|Weibrecht/Landegren 2012]]


#Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab
<ol>
#Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
<li>Antibody Activation</li>
#Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
<ol type="A">
#Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl
<li>Resuspend antibodies at 1 mg/mL in PBS</li>
#Store at 4C
<li>Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min</li>
<li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate</li>
<li>Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer</li>
<li>Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing</li>
<li>Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA</li>
<li>Transfer antibodies to new column and spin for 3 min at 1000xg</li></ol>
<li>Oligonucleotide Activation</li>
<ol type="A">
<li>Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA</li>
<li>Add 1.3 uL oligonucleotide to tube (or plate)</li>
<li>Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C</li>
<li>Add 20 uL PBS with 20 mM EDTA
<li>Remove excess DTT  using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS</li></ol>
<li>Ab-Oligo Mixing</li>
<ol type="A">
<li>Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab</li>
<li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li>
<li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li>
<li>Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl</li>
<li>Store at 4C</li>
</ol></ol>

Latest revision as of 17:57, 9 December 2016

Antibody-Oligo Linking[edit]

Back to Main

This page describes the protocol for antibody-oligo binding used in Combo-lock. The protocol is the first step in creating antibody probes. The antibodies are generally polyclonal antibodies from R&D systems. The protocol uses Sulfo-SMCC, which is a chemical that puts a reactive group on the terminal amine of the antibody. The oligos used have a thiol group that reacts with the Sulfo-SMCC activated antibody and links the oligonucleotide to the antibody.

Buffers[edit]

  1. Prep 100 mL of sodium phosphate buffer
    1. Make 100 mL of NaH2PO4 (monobasic) by adding 13.8g to nfH2O
    2. Make 100 mL of Na2H2PO4 (dibasic) by adding 14.2g to nfH2O
    3. Mix 28 mL of monobasic and 72 mL of dibasic to make 1 M (buffer pH 7.2)
    4. Directions from Cold Spring Harbor

Protocol[edit]

The protocol is taken (and slightly modified) from the Assarsson PEA Paper

Additional paper provides similar but more detailed protocol Weibrecht/Landegren 2012

  1. Antibody Activation
    1. Resuspend antibodies at 1 mg/mL in PBS
    2. Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min
    3. Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate
    4. Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer
    5. Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing
    6. Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA
    7. Transfer antibodies to new column and spin for 3 min at 1000xg
  2. Oligonucleotide Activation
    1. Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA
    2. Add 1.3 uL oligonucleotide to tube (or plate)
    3. Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C
    4. Add 20 uL PBS with 20 mM EDTA
    5. Remove excess DTT using two consecutive Zeba 7 kDa columns equilibrated with 100 mM PBS
  3. Ab-Oligo Mixing
    1. Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab
    2. Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
    3. Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
    4. Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl
    5. Store at 4C