MACS Matchmaker

NeutrAvidin / biotin capture — protocol

The NeutrAvidin DDI strategy uses NeutrAvidin (a deglycosylated avidin derivative with a near-neutral isoelectric point, $K_D \approx 10^{-14}\ \text{M}$ ) pre-conjugated to an oligo adapter. The NeutrAvidin-oligo hybridizes to any Oligo|PEG chip, then biotinylated ligands are captured via the effectively irreversible biotin–avidin bond. The chip is regenerable between different biotinylated ligands by stripping the DNA duplex. Validated on a range of biotinylated proteins and antibodies.

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

Both adapters ship as 110 µL stock at 2.6 µM in PBST — 10 immobilizations at the recommended 200 nM working concentration (130 µL/injection), Oligo|PEG chip (8×8 default; 6×9 on request), pre-conjugated to your chosen Seq ID (01–64) for multiplexing.

Adapter	Use	Webshop
Oligo Adapter NeutrAvidin	Tetrameric, effectively irreversible capture; use when the ligand must stay bound through harsh analyte or wash conditions	View in shop
Oligo Adapter Anti-Biotin	Monovalent anti-biotin VHH; the default adapter — reversible, one biotin per event, groove-matched on backfilled chips	View in shop

Strategy: adapter-first DDI capture

The NeutrAvidin-oligo conjugate is supplied ready-to-use. The experiment proceeds in two solution steps followed by two on-chip steps:

Four layers built bottom-up on the ridges. The NeutrAvidin–oligo conjugate hybridises to the chip strand, the biotinylated ligand loads onto the NeutrAvidin, and the analyte binds the ligand. Only the two on-chip steps are flow steps; the biotinylation happens in solution beforehand.

Biotinylation of the ligand (if needed). For non-biotinylated proteins, couple Biotin-PEG8-NHS to the primary amines in PBST pH 8.0. Use a spacer by default for proteins: without one, the biotin can sit too close to the protein surface for NeutrAvidin to reach it.
NeutrAvidin-oligo hybridization. Flow NeutrAvidin-oligo (200 nM in PBST) over the Oligo|PEG chip for 5 min. The oligo hybridizes to the complementary sequence on the chip ridges, loading the surface with NeutrAvidin binding sites.
Biotinylated ligand capture. Flow the biotinylated ligand at the desired concentration. Biotin binds NeutrAvidin with essentially irreversible kinetics under BIA conditions; surface loading is stable throughout the experiment.
Analyte measurement. Run the analyte series. Between different biotinylated ligands, strip the DNA duplex to reload NeutrAvidin fresh on the same chip.

Protocol

Step 1 — Biotinylation (non-biotinylated proteins only)

Parameter	Value
Reagent	Biotin-PEG8-NHS (10 mM stock in DMSO)
Molar ratio	1:3 protein : Biotin-PEG8-NHS
Working concentration	Dilute stock 100× in PBST pH 8.0 immediately before use
Buffer	PBST pH 8.0
Incubation	30–60 min, RT, 500 rpm
Quench and buffer exchange	Quench with 50 mM Tris pH 8.0 (10 min RT), then buffer-exchange into PBST using Amicon 10K or size-exclusion chromatography to remove excess biotin reagent

Step 2 — NeutrAvidin-oligo hybridization to chip

Parameter	Value
Adapter	NeutrAvidin-oligo on the Seq ID matching the chip strand
Concentration	200 nM in PBST
Flow rate	10 µL/min
Contact time	5 min
Chip type	Oligo\|PEG (any plex count)

Step 3 — Biotinylated ligand loading

Parameter	Value
Ligand concentration	10–100 nM biotinylated protein in PBST
Contact time	5–10 min (to saturation — monitor real-time signal)
Flow rate	10 µL/min

Step 4 — Analyte measurement and regeneration

Parameter	Value
Analyte buffer	PBST (match to ligand buffer)
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
After strip	Re-hybridize NeutrAvidin-oligo for the next ligand

Reference protocol parameters

Parameter	Reference value
Biotinylation reagent	Biotin-PEG8-NHS (spacer recommended by default for proteins)
Biotin : protein molar ratio	3 : 1
Biotinylation buffer	PBST pH 8.0, RT, 500 rpm, 30–60 min
NeutrAvidin-oligo concentration	200 nM
Hybridisation flow rate / contact time	10 µL/min, 5 min
Biotinylated ligand loading	10–100 nM, 10 µL/min, 5–10 min to saturation
Regeneration	3 M GuHCl + 125 mM NaOH, 400 µL/min, 0.5 min

QC and acceptance

Degree of biotinylation. Measure it — the HABA displacement assay, or the mass shift by intact-mass LC-MS — before the ligand reaches the chip. Under-biotinylated ligand is captured poorly and looks like a failed surface; over-biotinylated ligand binds several avidin sites at once, which immobilizes it in a constrained, multivalent way and changes the kinetics it reports. No validated biotins-per-molecule target applies across ligands, so establish the working value for yours and hold it constant across batches.
Capture succeeded. The ligand injection response persists through the wash, and an equivalent non-biotinylated ligand gives no such response.

The biotin bond is not selectively regenerated. It survives the conditions used to regenerate a binding surface, so analyte regeneration may leave the captured ligand in place. Changing the ligand requires stripping and reloading the complete oligo-adapter–ligand layer; there is no step that releases the ligand from the adapter while leaving the adapter behind.