Daniel:Protocols/AbOligoLink: Difference between revisions
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==Protocol== | ==Protocol== | ||
The protocol is taken (and slightly modified) from the [[Media:Assarsson_Homogenous_PEA_Immunoassay_PLOS_One_2014.PDF|Assarsson PEA Paper]] | |||
===Antibody Activation=== | ===Antibody Activation=== | ||
Revision as of 22:49, 26 May 2016
Antibody-Oligo Linking
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
- Resuspend antibodies at 1 mg/mL in PBS.
- 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
- 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 Zeba 7 kDa columns equilibrated with 100 mM PBS
Ab-Oligo Mixing
- Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab
- Transfer to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cup
- Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS
- 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
- Store at 4C