MACS Matchmaker
The AminoLink DDI strategy conjugates a protein's primary amines (lysines or N-terminus) to a TCO-functionalised oligonucleotide off-chip, then hybridises the resulting protein–oligo conjugate to any Oligo|PEG chip via DNA-Directed Immobilization (DDI). The duplex is the weakest link, so the chip is regenerable between different ligands by an alkaline strip. Validated on antibodies, Fabs, nanobodies, Fc receptors, and other recombinant proteins.
From the catalogue
Order both kit components from the lino Biotech webshop:
| Item | Contents | Webshop |
|---|---|---|
| Oligonucleotide Conjugation Kit, AminoLink | MTz-PEG-NHS bifunctional linker + purification reagents; supports 100 µg of protein conjugation; protein size range 15–300 kDa; total turnaround < 4 h | CHF 650 |
| Oligo Adapter for Amine Kit | Lyophilised, 10 nmol; 5′-TCO-modified ssDNA, one Seq ID (00–16) per vial; store at −20 °C | CHF 195 |
| Desalting Column, Zeba 7K | Buffer-exchange between coupling steps | CHF 21 |
| Spin Column, Amicon 10K | Final concentration / buffer exchange after IEX | CHF 21 |
Strategy: two-step off-chip coupling
Conjugation proceeds in two sequential reactions in solution, followed by ÄKTA purification to isolate the mono-conjugated fraction:
- MTz-PEG4-NHS linker coupling. The NHS ester reacts with a primary amine on the protein, installing a methyltetrazine (MTz) handle. A 1:2 protein-to-linker molar ratio is used to favour mono-labelling.
- TCO-oligo click. The MTz handle reacts with a TCO-functionalised oligo via inverse-electron-demand Diels–Alder (IEDDA). A 1:3 protein-to-oligo ratio drives the reaction to completion.
- ÄKTA IEX purification. Ion-exchange chromatography on a Resource Q column separates free protein, mono-conjugate, bi-conjugate, and free oligos. Only the mono-conjugated fraction is collected for use.
Protocol
Step 1 — Protein preparation
Rehydrate lyophilised protein per the manufacturer specification. For liquid stocks, centrifuge at 14,000 rpm, 5 min, RT to pellet aggregates. Measure concentration by NanoDrop (A280, using the molar extinction coefficient ε₂₈₀ for the specific protein).
Step 2 — MTz-PEG4-NHS linker coupling
| Parameter | Value |
|---|---|
| Linker | MTz-PEG4-NHS (100 mM stock in DMSO) |
| Molar ratio | 1:2 protein : linker |
| Working linker concentration | 1 mM (dilute stock 100× in PBST pH 8.0) |
| Buffer | PBST pH 8.0 |
| Incubation | 1–2.5 h, 25 °C, 500 rpm |
Step 3 — TCO-oligo click
| Parameter | Value |
|---|---|
| Oligo | TCO-oligo (cs01, cs02, … — chip-sequence matched) |
| Oligo stock | 100–500 µM in PBST pH 7.4 |
| Molar ratio | 1:3 protein : oligo |
| Incubation | 1.5 h, 25 °C, 500 rpm |
Step 4 — ÄKTA IEX purification
Load the reaction mixture onto a Resource Q column (IEX). Use a salt gradient (Buffer A: low salt, Buffer B: high salt) with a 2 CV elution. Peaks elute in order:
- Non-conjugated protein (peaks A and B)
- Mono-conjugated protein–oligo (peak C) — collect this fraction
- Bi- and tri-conjugates (peaks D, E)
- Free oligos and poly-conjugates (peak F)
Typical yield: ~20% mono-conjugated from recovered protein, ~93% total protein recovery from the column.
Step 5 — Buffer exchange and concentration
Pool the mono-conjugate fractions. Buffer-exchange into PBST using an Amicon 10K 2 mL ultrafiltration unit (7,000×g, 7 min, 5 × PBST washes). Measure final concentration by NanoDrop; the 260/280 ratio should shift above 1.2 confirming oligo attachment.
Step 6 — Hybridisation to chip and measurement
| Parameter | Value |
|---|---|
| Ligand concentration | 200 nM in PBST |
| Flow rate | 10 µL/min |
| Contact time | 5 min |
| Chip type | Oligo|PEG (any plex count) |
| SCK association flow rate | 30 µL/min (6-ch) / 60 µL/min (3–4-ch) / 100 µL/min (1-ch) |
| SCK dissociation flow rate | 200 µL/min |
| Regeneration (duplex strip) | 3 M guanidinium-HCl + 125 mM NaOH, 400 µL/min, 0.5 min |
Optimal parameters at a glance
| Parameter | Optimal value |
|---|---|
| Linker : protein molar ratio | 2:1 |
| Linker coupling buffer | PBST pH 8.0, 25 °C, 500 rpm, 1–2.5 h |
| Oligo : protein molar ratio | 3:1 |
| Oligo coupling conditions | PBST pH 7.4, 25 °C, 500 rpm, 1.5 h |
| Purification column | Resource Q (IEX), 2 CV elution |
| Final buffer | PBST, Amicon 10K buffer exchange |
| Hybridisation concentration | 200 nM |
| Hybridisation flow rate | 10 µL/min, 5 min |
| SCK association flow rate | 30 µL/min (6-ch) / 60 µL/min (3–4-ch) / 100 µL/min (1-ch) |
| SCK dissociation flow rate | 200 µL/min |
| Regeneration | 3 M GuHCl + 125 mM NaOH, 400 µL/min, 0.5 min |
Practical notes
See also: immobilisation method comparison table.