The Anti-Biotin DDI strategy uses a monovalent anti-biotin VHH (single-domain antibody) pre-conjugated to an oligo adapter. The Anti-Biotin-oligo hybridizes to any Oligo|PEG chip, then biotinylated ligands are captured one biotin per binding event — no avidity cross-linking, so kinetics are read cleanly whatever the degree of biotinylation. The capture is dissociable, which keeps the ridge and groove chemistries matched on a backfilled chip, and this is the default biotin-capture adapter. Use NeutrAvidin instead when the ligand must stay captured through conditions that would elute an antibody.
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
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Oligo Adapter Anti-Biotin
Monovalent anti-biotin VHH; default choice — reversible capture, one biotin per event, groove-matched on backfilled chips
The Anti-Biotin-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 Anti-Biotin–oligo conjugate hybridises to the chip strand, the biotinylated ligand loads onto the anti-biotin VHH, 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 the anti-biotin VHH to reach it.
Anti-Biotin-oligo hybridization. Flow Anti-Biotin-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 anti-biotin binding sites.
Biotinylated ligand capture. Flow the biotinylated ligand at the desired concentration. The VHH binds one biotin per site; capture is stable for the measurement yet dissociable for regeneration.
Analyte measurement. Run the analyte series. Between different biotinylated ligands, strip the DNA duplex to reload the adapter fresh on the same chip.
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 — Anti-Biotin-oligo hybridization to chip
Parameter
Value
Adapter
Anti-Biotin-oligo on the Seq ID matching the chip strand
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)
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)
Re-hybridize Anti-Biotin-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
Anti-Biotin-oligo concentration
200 nM
Hybridisation flow rate / contact time
10 µL/min, 5 min, ambient
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. Monovalent capture tolerates several biotins per molecule without the avidity distortion a tetrameric adapter would show, but hold the working value constant across batches so surface density stays comparable.
Capture succeeded. The ligand injection response persists through the wash, and an equivalent non-biotinylated ligand gives no such response.
Capture is stable enough for the run. Because the anti-biotin bond is dissociable, check the pre-analyte baseline is flat over a hold comparable to your dissociation phase; a slow downward drift is ligand leaving the adapter, not analyte kinetics.