MACS Matchmaker

NeutrAvidin / biotin capture — protocol

The NeutrAvidin DDI strategy uses NeutrAvidin (deglycosylated streptavidin, KD≈10−14 MK_D \approx 10^{-14}\ \text{M}) pre-conjugated to an oligo adapter. The NeutrAvidin-oligo hybridises 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.

From the catalogue

Both adapters ship as 110 µL stock at 2.6 µM in PBST — 10 immobilisations 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 (00–16) for multiplexing.

AdapterUseWebshop
Oligo Adapter NeutrAvidinTetrameric capture of biotinylated ligands; default choice for a single biotin per ligandCHF 195
Oligo Adapter Anti-BiotinMonovalent anti-biotin VHH; use for multi-biotinylated ligands where avidity cross-linking would distort kineticsCHF 214.50

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:

  1. Biotinylation of the ligand (if needed). For non-biotinylated proteins, couple Biotin-PEG8-NHS to the primary amines in PBST pH 8.0. A PEG8 spacer is essential for proteins above ~28 kDa to prevent steric occlusion of the biotin by NeutrAvidin.
  2. NeutrAvidin-oligo hybridisation. Flow NeutrAvidin-oligo (200 nM in PBST) over the Oligo|PEG chip for 5 min. The oligo hybridises to the complementary sequence on the chip ridges, loading the surface with NeutrAvidin binding sites.
  3. 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.
  4. 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)

ParameterValue
ReagentBiotin-PEG8-NHS (10 mM stock in DMSO)
Molar ratio1:3 protein : Biotin-PEG8-NHS
Working concentrationDilute stock 100× in PBST pH 8.0 immediately before use
BufferPBST pH 8.0
Incubation30–60 min, RT, 500 rpm
Quench and buffer exchangeQuench 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 hybridisation to chip

ParameterValue
AdapterNeutrAvidin-oligo (cs01, cs02, … — chip-sequence matched)
Concentration200 nM in PBST
Flow rate10 µL/min
Contact time5 min
Chip typeOligo|PEG (any plex count)

Step 3 — Biotinylated ligand loading

ParameterValue
Ligand concentration10–100 nM biotinylated protein in PBST
Contact time5–10 min (to saturation — monitor real-time signal)
Flow rate10 µL/min

Step 4 — Analyte measurement and regeneration

ParameterValue
Analyte bufferPBST (match to ligand buffer)
SCK association flow rate30 µL/min (6-ch) / 60 µL/min (3–4-ch) / 100 µL/min (1-ch)
SCK dissociation flow rate200 µL/min
Regeneration (duplex strip)3 M guanidinium-HCl + 125 mM NaOH, 400 µL/min, 0.5 min
After stripRe-hybridise NeutrAvidin-oligo for the next ligand

Optimal parameters at a glance

ParameterOptimal value
Biotinylation reagentBiotin-PEG8-NHS (PEG8 spacer required for proteins >28 kDa)
Biotin : protein molar ratio3 : 1
Biotinylation bufferPBST pH 8.0, RT, 500 rpm, 30–60 min
NeutrAvidin-oligo concentration200 nM
Hybridisation flow rate / contact time10 µL/min, 5 min
Biotinylated ligand loading10–100 nM, 10 µL/min, 5–10 min to saturation
Regeneration3 M GuHCl + 125 mM NaOH, 400 µL/min, 0.5 min

Practical notes

See also: immobilisation method comparison table.