Daniel:Protocols/AbOligoLink: Difference between revisions

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#Add 1.3 uL oligonucleotide to tube (or plate)
#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 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
#Add 20 uL PBS with 20 mM EDTA  
#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
#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



Revision as of 17:41, 14 October 2016

Antibody-Oligo Linking

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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

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

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
    1. 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

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

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