MACS Matchmaker
The ThioLink DDI strategy selectively couples a free or engineered cysteine on a protein to a maleimide-functionalised oligonucleotide off-chip, then hybridises the resulting protein–oligo conjugate to any Oligo|PEG chip via DNA-Directed Immobilization (DDI). Because only accessible cysteines react, orientation is more reproducible than random amine coupling. Validated on nanobodies and other single-Cys proteins.
From the catalogue
Order both kit components from the lino Biotech webshop:
| Item | Contents | Webshop |
|---|---|---|
| Oligonucleotide Conjugation Kit, ThioLink | TCEP, maleimide-coupling buffers, purification reagents; supports ~100 µg of protein conjugation (e.g. VHH); total turnaround < 5 h | CHF 650 |
| Oligo Adapter for Thiol Kit | Lyophilised, 10 nmol; 5′-maleimide-modified ssDNA, one Seq ID (00–16) per vial; store at −20 °C | CHF 195 |
| Desalting Column, Zeba 7K | Buffer-exchange after TCEP reduction | CHF 21 |
| Spin Column, Amicon 10K | Final concentration / buffer exchange after IEX | CHF 21 |
Strategy: TCEP reduction then maleimide click
Conjugation proceeds in two sequential reactions in solution, with an optional pre-purification step for His-tagged proteins, followed by ÄKTA IEX to isolate the mono-conjugated fraction:
- TCEP reduction. A mild reducing agent (tris(2-carboxyethyl)phosphine, TCEP) breaks any disulfide bonds and generates free thiols. A 1.5-fold molar excess of TCEP over protein is used to minimise over-reduction artefacts.
- Optional HisTrap pre-purification. For His-tagged proteins, pass the reduced sample over a HisTrap column to remove TCEP and buffer-exchange before maleimide coupling. Skip for proteins without a His-tag.
- Maleimide-oligo click. The free thiol reacts with a maleimide-functionalised oligo to form a stable thioether bond. A 1:3 protein-to-oligo ratio drives the reaction to completion, and unreacted thiols are quenched with N-ethylmaleimide (NEM).
- ÄKTA IEX purification. Ion-exchange chromatography on a Resource Q column separates free protein, mono-conjugate, and bi-conjugates. Only the mono-conjugated fraction is collected.
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). Dilute to 0.5 mg/mL in PBS + 500 mM NaCl.
Step 2 — TCEP reduction
| Parameter | Value |
|---|---|
| Reductant | TCEP (tris(2-carboxyethyl)phosphine) |
| Molar ratio | 1.5 eq TCEP : 1 eq protein |
| Working concentration | 0.5 mg/mL protein in PBS + 500 mM NaCl |
| Incubation | 1 h, RT, 800 rpm (pipette to mix — do not vortex) |
Step 3 — Optional HisTrap pre-purification (His-tagged proteins)
Load the TCEP-reduced protein onto a HisTrap HP column equilibrated with binding buffer (PBS + 500 mM NaCl + 20 mM imidazole). Wash with 5 CV binding buffer, then elute with 250 mM imidazole. This removes TCEP and exchanges the buffer before maleimide coupling. Skip for non-His-tagged proteins.
Step 4 — Maleimide-oligo conjugation
| Parameter | Value |
|---|---|
| Oligo | Maleimide-oligo (cs01, cs02, … — chip-sequence matched) |
| Oligo stock | 100–500 µM in PBS pH 7.0 |
| Molar ratio | 1:3 protein : oligo |
| Incubation | 1–2 h, RT, 500 rpm |
| Quench unreacted thiols | Add N-ethylmaleimide (NEM) to 10-fold molar excess over TCEP; incubate 15 min, RT |
Step 5 — 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: ~25–40% mono-conjugated from recovered protein.
Step 6 — 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 7 — 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 |
|---|---|
| Protein working concentration | 0.5 mg/mL in PBS + 500 mM NaCl |
| TCEP : protein molar ratio | 1.5 : 1 |
| TCEP reduction conditions | PBS + 500 mM NaCl, RT, 800 rpm, 1 h (pipette to mix) |
| Oligo : protein molar ratio | 3 : 1 |
| Maleimide coupling conditions | PBS pH 7.0, RT, 500 rpm, 1–2 h |
| Thiol quench | 10× molar excess NEM over TCEP, 15 min RT |
| 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.