MACS Matchmaker
The ThioLink DDI strategy couples the accessible cysteine of a VHH antibody to a maleimide-functionalized oligonucleotide off-chip with the Oligonucleotide Conjugation Kit, ThioLink, then hybridizes the resulting VHH–oligo conjugate to any Oligo|PEG chip via DNA-Directed Immobilization (DDI). Because only the accessible thiol reacts, orientation is more reproducible than random amine coupling. The kit is designed for His-tagged VHH antibodies: the free oligo is removed on a Ni-NTA column that retains the conjugate by its tag.
Before you start
- Read Safety & Compliance and the safety data sheet for every reagent below. The SDS, not this page, governs how a reagent is handled, stored and disposed of.
- Use the personal protective equipment and the waste route your laboratory prescribes for these reagent classes.
- Check this protocol against the materials actually in hand: confirm the intended Seq ID, the product revision, the lot-specific certificate and the instructions supplied with it. Where those differ from this page, the documentation supplied with the material governs.
From the catalogue
Order the kit and the matching adapter from the lino Biotech webshop:
| Item | Contents | Webshop |
|---|---|---|
| Oligonucleotide Conjugation Kit, ThioLink | Protein Preparation Buffer, 10× reducing reagent, Ni-NTA column with elution buffer, two protein concentrators and collection tubes; one conjugation of 100 µg of VHH antibody in under 5 h | View in shop |
| Oligo Adapter for Thiol Kit | Lyophilised, 10 nmol; 5′-maleimide-modified ssDNA, one Seq ID (01–64) per vial; reconstitute at 100 µM; store at −20 °C | View in shop |
| Spin Column, Amicon 10K | Spare protein concentrator for the two buffer-exchange steps; two are included in the kit | View in shop |
Strategy: reduction then maleimide click
Conjugation proceeds in two reactions in solution, each followed by a clean-up, with the His-tag doing the work of separating conjugate from free oligo:
- Reduction. The kit's reducing reagent opens the accessible disulfide and frees the cysteine thiol.
- Removal of the reducing reagent. A buffer exchange on the protein concentrator takes the reductant out before the maleimide is added, so nothing competes with the cysteine.
- Maleimide-oligo click. The free thiol reacts with the maleimide-functionalized oligo to form a stable thioether bond.
- Removal of free oligo. The His-tagged conjugate binds the Ni-NTA column while free oligo washes through; imidazole elutes the conjugate.
- Removal of imidazole. A second buffer exchange on the protein concentrator leaves the conjugate in Protein Preparation Buffer at about 100 µM.
Protocol
Step 1 — Protein requirements
Start from 100 µg of VHH antibody at 1 mg/mL, free of carrier proteins with accessible thiols or disulfide bridges and free of thiol reductants. Mix by pipetting throughout: VHH antibodies may precipitate when vortexed or centrifuged at high speed.
Step 2 — Reduce the VHH antibody
Spin down the vial of 10× reducing reagent and add 450 µL Protein Preparation Buffer to it. Add 50 µL of the diluted reagent to the solution containing 100 µg of VHH antibody, mix by pipetting and incubate in a thermomixer at 25 °C and 500 rpm for 1 h.
Step 3 — Remove the reducing reagent
Wash a protein concentrator with 500 µL Protein Preparation Buffer at 14,000 × g for 10 min, with the cap strap and one membrane panel facing the centre of the rotor. Bring the reduced antibody to 450 µL with Protein Preparation Buffer, load it and spin at 14,000 × g for 10 min. Discard the flow-through, refill with 450 µL buffer and spin again; repeat the refill once more. Invert the concentrator into a fresh collection tube and spin at 1,000 × g for 2 min; about 50 µL at about 2 mg/mL is recovered.
Step 4 — Conjugate to the maleimide-oligo
Reconstitute the adapter at 100 µM in nuclease-free water, PBS or Protein Preparation Buffer. Add 300 µL of it to the reduced antibody and incubate in a thermomixer at 25 °C and 500 rpm for 1 h.
Step 5 — Remove free oligo on the Ni-NTA column
Remove the bottom closure of the Ni-NTA column and keep it; loosen the cap. In a 2 mL collection tube, spin at 700 × g for 2 min to remove the storage solution, then equilibrate with 400 µL Protein Preparation Buffer at 700 × g for 2 min, three times. Close the bottom, apply the conjugation mixture to the centre of the resin and incubate in a thermomixer at 25 °C and 500 rpm for 30 min. Remove the closure and spin at 700 × g for 2 min, then wash with 200 µL Protein Preparation Buffer at 700 × g for 2 min, five times in all. Move the column to a fresh collection tube and elute with 200 µL Ni-NTA Elution Buffer at 700 × g for 2 min, three times, combining the eluates (600 µL).
Step 6 — Remove imidazole
Wash the second protein concentrator with 500 µL Protein Preparation Buffer at 14,000 × g for 10 min. Load 300 µL of the eluate, spin at 14,000 × g for 10 min and discard the flow-through; load the remaining 300 µL and spin again. Refill with 450 µL Protein Preparation Buffer and spin at 14,000 × g for 10 min, twice. Invert into a fresh collection tube and spin at 1,000 × g for 2 min. About 50 µL of conjugate at about 1.5 mg/mL (100 µM) is recovered; roughly a quarter of the antibody is lost across the purification steps.
Aliquot and store at +4 °C for up to 3 months.
Step 7 — Hybridisation to chip and measurement
| Parameter | Value |
|---|---|
| Ligand concentration | 200 nM in PBST |
| Flow rate | 10 µL/min |
| Contact time | 5 min |
| Temperature | Ambient (the reader holds no fixed set point) |
| 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 GuHCl + 125 mM NaOH, 400 µL/min, 0.5 min |
Reference protocol parameters
| Parameter | Reference value |
|---|---|
| Protein input | 100 µg VHH antibody at 1 mg/mL, no thiol reductants |
| Reduction | 50 µL of 1× reducing reagent, 25 °C, 500 rpm, 1 h |
| Reductant removal | Protein concentrator, 14,000 × g, 10 min, two buffer refills |
| Conjugation | 300 µL maleimide-oligo at 100 µM, 25 °C, 500 rpm, 1 h |
| Free-oligo removal | Ni-NTA column, 30 min binding, five 200 µL washes, three 200 µL imidazole elutions |
| Imidazole removal | Protein concentrator, 14,000 × g, 10 min, two buffer refills |
| Yield | About 50 µL at about 100 µM; about 25 % of the antibody lost |
| Storage of the conjugate | +4 °C, up to 3 months |
| 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 |
QC and acceptance
Two failures bracket this chemistry: too little reduction leaves no free thiol for the maleimide, and too much takes the ligand apart. Both are checked before coupling, not after.
- Free thiol available. Quantify free thiols after reduction — Ellman's reagent (DTNB) against a cysteine standard is the usual assay — and compare the thiols per molecule with the number the construct should expose. A result near zero means the reduction did not work, or the thiols reoxidized before the assay.
- Not over-reduced. A VHH has no interchain disulfides to shed, so over-reduction shows as lost activity rather than fragments: its single intradomain disulfide opens and the domain unfolds. The kit fixes the reduction step, so if activity is lost, look for extra reductant in the protein formulation rather than shortening the step.
- Still active. A ligand can survive both checks and have lost its binding site to a reduced structural disulfide. Confirm activity against a known partner before committing chip time.
How many thiols a construct exposes under the kit's fixed reduction is construct-specific and is not validated here. Determine it once for your VHH.
Practical notes
See also: immobilization method comparison table.