MACS Matchmaker

Off-chip TCO activation of amine-bearing ligands — protocol

For direct, covalent immobilization on a [Me-Tz|PEG] or [Me-Tz|Tz] sensor, the ligand has to carry a TCO group. This page describes the off-chip activation route: a bifunctional NHS–PEG–TCO linker reacts with the ligand's primary amines (N-terminal or lysine side-chains), leaving a TCO handle on the ligand that then clicks onto surface tetrazines via inverse-electron-demand Diels–Alder (IEDDA). The product is a covalent, oriented-once TCO-tagged ligand ready for flow injection.

Strategy
Linker chemistry choice
Protocol
Optimal parameters
Practical notes

Strategy

A bifunctional NHS–PEG–TCO linker is reacted with the ligand at a controlled molar ratio. The NHS end couples to a surface lysine (or the N-terminus); the TCO end is left exposed as a click handle. After removing free / hydrolysed linker on a small desalting column, the TCO-tagged ligand is injected onto a Me-Tz sensor and clicks onto the ridges in seconds.

TCO activation process: NHS coupling then IEDDA click on chip ridge — ① The NHS ester of the TCO–PEG₄–NHS linker reacts with a primary amine on the protein, installing a TCO handle. ② The TCO-tagged protein is purified. ③ On injection, TCO clicks covalently onto MeTz / Tz groups on the chip ridge via inverse-electron-demand Diels–Alder (IEDDA).

Linker chemistry choice

Three amine-reactive ester chemistries are commonly available for PEG–TCO linkers. The trade-off is between aqueous stability and commercial availability:

Active group	Aqueous stability	Solubility	Notes
NHS	Hydrolyses fastest	Good	Most widely available — the practical default. Use freshly prepared semi-stock and add immediately.
TFP	Stable	Lower	Higher coupling yield than NHS; lower solubility can make downstream purification trickier.
STP	Most stable	Good	Best control but PEGylated TCO–STP variants are not consistently in catalogue.

The protocol below assumes the most accessible variant, TCO–PEG4–NHS (100 mM stock in anhydrous DMSO).

TCO–PEG4–NHS linker structure — TCO–PEG4–NHS linker: trans-cyclooctene (left) connected via a four-unit PEG spacer to the NHS ester (right)

Protocol

Step 1 — Buffer exchange (amine-free buffer)

NHS chemistry requires the absence of competing amines. If the ligand is in Tris, glycine, histidine, or any other amine-bearing buffer, exchange into PBS-T (pH 7.4) using a 10 K Amicon spin column (3 K for ligands < 20 kDa). Four wash cycles at 14.1 k rcf for 10 min each are usually enough; a final wash in PBS-T pH 8.0 prepares the sample for the activation step.

Step 2 — Confirm the ligand concentration

Measure the ligand concentration on a NanoDrop (or equivalent UV A280) blanking against PBS-T pH 8.0. Take the median of three readings. Convert to molarity using the ligand's molecular weight — the linker stoichiometry depends on it.

Step 3 — Activation reaction (1:2 ligand:linker)

Parameter	Value
Linker	TCO–PEG4–NHS, 100 mM stock in anhydrous DMSO
Molar ratio	1 : 2 (ligand : linker)
Buffer	PBS-T pH 8.0
Reaction	25 °C, 1 h, 500 rpm orbital shaking
Working semi-stock	Dilute 2 µL of 100 mM TCO–PEG4–NHS in PBS-T pH 8.0 to give the volume calculated for your ligand amount; add the 2 µL semi-stock immediately to the ligand and mix vigorously.

Step 4 — Remove free linker (Zeba 7K, two passes)

Pass the activated ligand twice through fresh Zeba 7K spin desalting columns (300 µL PBS-T pH 7.4 wash steps; load 120 µL sample; spin 1.5 k rcf, 2 min). Two passes consistently remove > 95 % of free linker / hydrolysis products. The first column pass also brings the ligand into pH 7.4, the storage buffer.

Step 5 — Final concentration and aliquoting

Re-measure the concentration on the NanoDrop (blanked against PBS-T pH 7.4). Aliquot the activated ligand at the highest concentration possible, snap-freeze, and store at −20 °C. Avoid freeze–thaw cycles. Typical aliquot sizes are 0.2–0.5 nmol per tube for Tz-sensor use, 1.0–1.5 nmol for Me-Tz-sensor use.

Optimal parameters at a glance

Parameter	Optimal value
Linker	TCO–PEG4–NHS
Linker stock	100 mM in anhydrous DMSO
Activation buffer	PBS-T pH 8.0 (amine-free)
Ligand : linker ratio	1 : 2 mol
Reaction	25 °C, 1 h, 500 rpm
Purification	Zeba 7K desalting × 2 (PBS-T pH 7.4)
Storage	−20 °C, single-use aliquots, no freeze–thaw
On-chip injection	1–10 µM, copper-free, seconds-to-minutes

Practical notes

Once activated, inject the TCO-tagged ligand onto a [Me-Tz|PEG] (clean-buffer) or [Me-Tz|Tz] (matrix-tolerant) sensor. The chip is single-use per ligand — covalent capture is irreversible. For the full chip-and-adapter context, return to the interactive decision tree — the direct TCO–tetrazine click branch lands here.