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:

Strategy: two-step off-chip coupling

Conjugation proceeds in two reactions in solution, each followed by a clean-up on a kit column:

1. Me-Tz linker activation. The NHS ester of the bifunctional linker reacts with a primary amine on the protein, installing a methyltetrazine (Me-Tz) handle. The linker volume sets the average number of handles per protein, from one to five.
2. Removal of excess linker. The desalting column retains free linker, which would otherwise consume TCO-oligo in the next step.
3. TCO-oligo click. The Me-Tz handle reacts with the TCO-functionalized oligo via inverse-electron-demand Diels–Alder (IEDDA). The oligo volume follows the same protein-to-oligo ratio.
4. Removal of free oligo. The oligonucleotide capturing slurry retains free oligo while the conjugate passes through.

Protocol

Step 1 — Protein requirements and optional buffer exchange

Start from 100 µg of protein (15–300 kDa) at 1–5 mg/mL, free of carrier proteins such as BSA. The NHS ester reacts with any primary amine, so the formulation must meet the requirements below; exchange the buffer first if it does not, or if the protein is below 1 mg/mL. Reconstitute lyophilised protein at 1 mg/mL in Protein Preparation Buffer.

To exchange the buffer, wash the protein concentrator with 500 µL Protein Preparation Buffer at 14,000 × g for 10 min, bring the protein to 450 µL with Protein Preparation Buffer, spin at 14,000 × g for 10 min and discard the flow-through, then refill with 450 µL buffer and spin again. Repeat the refill as many times as the amine-containing component needs: the number of exchanges is the decimal logarithm of its concentration in µM, rounded up (20 mM Tris needs five). 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 2 — Reconstitute the linker

Add 10 µL Linker Reconstitution Medium to the bifunctional linker and pipette vigorously until the red pellet has dissolved. Add 2.0 mL Protein Preparation Buffer, vortex for at least 5 s and spin down. Use the solution immediately.

Step 3 — Activate the protein

Add the linker to the solution containing 100 µg of protein. The volume follows from the protein's molecular weight and the number of oligos wanted per protein:

linker volume (µL) = oligos per protein ÷ molecular weight (kDa) × 1000

Incubate in a thermomixer at 25 °C and 800 rpm for 1 h. The ratio is a statistical average: most proteins carry the chosen number of handles, individual molecules carry more or fewer.

Step 4 — Remove excess linker

Remove the bottom closure of the desalting column, loosen its cap, place it in a 2 mL collection tube and spin at 1,500 × g for 1 min to remove the storage solution. Mark the side where the resin slants upward and keep that mark facing outward in every following spin. Equilibrate with 300 µL Protein Preparation Buffer at 1,500 × g for 1 min, three times. Bring the activated protein to 130 µL with Protein Preparation Buffer, apply it slowly to the centre of the resin and collect it at 1,500 × g for 2 min.

Step 5 — Conjugate to the TCO-oligo

Reconstitute the adapter at 100 µM in nuclease-free water, PBS or Protein Preparation Buffer. Add it to the purified activated protein; the volume follows the same ratio as the linker:

oligo volume (µL) = oligos per protein ÷ molecular weight (kDa) × 1500

Incubate in a thermomixer at 25 °C and 800 rpm for 1 h.

Step 6 — Remove free oligo

Vortex the oligonucleotide capturing slurry briefly and add 200 µL to the spin column. In a cap-free collection tube, spin at 700 × g for 2 min, then equilibrate with 200 µL Protein Preparation Buffer at 700 × g for 2 min, three times. Bring the conjugation mixture to 200 µL with Protein Preparation Buffer, close the bottom of the spin column, place it in a fresh 2 mL collection tube and apply the sample slowly to the centre of the slurry. Incubate in a thermomixer at 25 °C and 800 rpm for 30 min. Remove the bottom closure and spin at 700 × g for 2 min; add 200 µL Protein Preparation Buffer and spin again to recover the rest. The collection tube now holds the purified conjugate.

The final concentration depends on the protein and the ratio used; measure it with an appropriate assay where it matters. Aliquot and store at +4 °C for up to 3 months.

Step 7 — Hybridisation to chip and measurement

Reference protocol parameters

QC and acceptance

Before the conjugate goes on a chip, establish that the ligand is actually carrying oligo. The conjugation is the step that fails silently: an unconjugated ligand behaves exactly like a ligand that never bound its target.

• Off-chip. A mobility shift against the unconjugated ligand on SDS-PAGE, or the corresponding shift by SEC, shows that oligo is attached. An A₂₆₀/A₂₈₀ ratio raised above the ligand's own value is consistent with the same thing and is quicker, but it does not distinguish attached oligo from free oligo carried through the purification.
• On-chip. The conjugate injection gives an immobilization response that survives the following buffer wash, and a mologram carrying a non-complementary strand does not. That pair separates hybridization from adsorption.

There is no validated degree-of-conjugation threshold for this protocol. Record the value you measure and keep it comparable across batches rather than judging it against a number.

Practical notes

See also: immobilization method comparison table.