Manuals+

helix+ User Manual

NEW Y-STRUCTURE KIT 2

for proximity binding assay - Spot 2

Dynamic Biosensors GmbH, HK-NYS-2 v1.0

Key Features

  • Ideal for studying ternary complex formation upon binding of bispecific small molecules (e.g., PROTACs, molecular glues), homo-dimerization, and bispecific antibodies with weak affinities.
  • The new optimized Y-structure design allows higher FRET sensitivity.
  • Compatible with heliX® Adapter Chip.
  • The Y-structure Red Adapter strand (specific to Spot 2) carries a moderately hydrophilic red dye (Ra) with a single positive net charge.
  • The Y-structure Green Adapter strand carries a hydrophilic green dye (Ga) with a single negative net charge.
  • Green and red dyes can detect binding on each arm via Fluorescence Proximity Sensing (FPS), or report if the structure is OPEN or CLOSED via sensitive FRET.
  • Homo-/hetero-proteins can be coupled easily to the arms via exchangeable ligand strands.
  • The flexible hinge region confines two proteins to a small volume and defined distance.

Product Description

Order Number: HK-NYS-2

Table 1. Contents and Storage Information

Material Cap Concentration Amount Buffer Storage
New Y-structure Red Adapter strand 2 with Ra Orange 400 nM 5 x 50 µL TE40 [1] -20°C
New Y-structure Green Adapter strand with Ga Green 400 nM 5 x 50 µL TE40 [1] -20°C
New Y-structure Ligand Free Strand (Ifs) - Red for binary binding in green Yellow 500 nM 3 x 100 µL TE40 [1] -20°C
New Y-structure Ligand Free Strand (Ifs) - Green for binary binding in red Yellow 500 nM 3 x 100 µL TE40 [1] -20°C

For research use only. This product has a limited shelf life, please see expiry date on label. To avoid many freeze thaw cycles please aliquot the nanolever.

[1] TE40: 10 mM Tris, 40 mM NaCl, 0.05% Tween20, 50 µM EDTA, 50 µM EGTA

heliX® Adapter Chip Overview

Ternary binding

This configuration involves an anchor strand on Spot 1, and on Spot 2, an anchor strand with both a Red Adapter strand and a Green Adapter strand. The Red Adapter strand is conjugated with Ligand 1 (Red dye), and the Green Adapter strand is conjugated with Ligand 2 (Green dye). This setup is for studying ternary complex formation.

Binary binding in green

This configuration uses an anchor strand on Spot 1. On Spot 2, an anchor strand is attached to a Red Adapter strand and a Green Adapter strand. The Red Adapter strand is free of ligand (Ligand-Free strand 1 - Red), while the Green Adapter strand is conjugated with Ligand 2 (Green dye). This setup is for binary binding detection where the green signal is primary.

Binary binding in red

Similar to the above, but the roles are reversed. On Spot 2, the Red Adapter strand is conjugated with Ligand 1 (Red dye), and the Green Adapter strand is ligand-free (Ligand-Free strand 2 - Green). This setup is for binary binding detection where the red signal is primary.

Preparation

The ligand strands can be extended at either the 5' (Ligand strand 1 - Red arm) or 3' (Ligand strand 2 - Green arm) end with any DNA/RNA sequence. They can also be crosslinked to a protein of interest through amine coupling using specialized heliX® Amine Coupling Kits (HK-NYS-NHS-1 for the red arm, HK-NYS-NHS-2 for the green arm). Purification of the conjugation product with proFIRE® is highly recommended before conducting kinetic studies.

TIP: For higher FRET quality, covalent coupling of ligands is recommended. Alternatively, proteins can be captured via His-tag using the HK-NYS-NTA kit.

Automated Sample Preparation Workflow

The heliX® instrument performs automated functionalization and regeneration of the chip. A mobile app, helix® MIX&RUN, is available for use on mobile devices.

The general steps involve:

  • Step 1: Couple Ligands: Covalently attach ligands to ligand strands (e.g., via NHS coupling). Use ready-to-use coupling kits.
  • Step 2: Select Adapter: Choose appropriate ready-to-use adapter strands.
  • Step 3: Mix: Combine ligand strands and adapter strands. For example, mixing 100 nM ligands with 90 nM Y-Structure strands aims for a final concentration of 50 nM.
  • Step 4: Functionalization: The heliX® instrument handles automated functionalization and regeneration.

Example Hybridization Protocol

This example details the preparation for measuring ternary binding on Spot 1 and binary binding in green on Spot 2.

Vial 1 (for Spot 1): In solution hybridization of Y-structure strands and ligand strands (not included in this kit; refer to HK-NYS-1, HK-NYS-NHS-1, and HK-NYS-NHS-2).

  1. Mix Y-structure Red Adapter strand (400 nM), Green Adapter strand (400 nM), conjugated Ligand strand 1 (500 nM), and conjugated Ligand strand 2 (500 nM) at a 1:1 ratio (v/v).
  2. Incubate the solution at room temperature (RT) for 2 hours to ensure complete hybridization. Overnight incubation at 4°C is also possible, depending on the stability of the conjugated protein.

Vial 2 (for Spot 2): In solution hybridization of Y-structure strands and ligand strands.

  1. Mix Y-structure Red Adapter strand 2 (400 nM), Green Adapter strand (400 nM), Ligand-free strand 1 - Red (500 nM), and conjugated Ligand strand 2 - Green (500 nM) at a 1:1 ratio (v/v).
  2. Incubate the solution at RT for 2 hours to ensure complete hybridization. Overnight incubation at 4°C is also possible, depending on the stability of the conjugated protein.

Step 3: Mixing
Mix solution of Vial 1 and Vial 2 at a 1:1 ratio (v/v).

Step 4: Ready for Use
The resulting solution is ready for heliX® Adapter Chip functionalization.

Example Volumes for Functionalization

Required volume for 1 functionalization: 35 µL with a final concentration of 50 nM.

Preparation Step Component Volume
Vial 1 New Y-structure Red Adapter strand & Green Adapter strand (400 nM) 4.5 µL each > 9 µL total
Conjugated Ligand strand 1 & Conjugated Ligand strand 2 (500 nM) 4.5 µL each > 9 µL total
Vial 2 New Y-structure Red Adapter strand 2 & Green Adapter strand (400 nM) 4.5 µL each > 9 µL total
Ligand-free strand 1 - Red & Conjugated Ligand strand 2 (500 nM) 4.5 µL each > 9 µL total

After incubation, mix Vial 1 and Vial 2 to obtain the ready-to-use DNA solution.

Assay Setup in heliOS

For studying ternary complex formation upon binding of bispecific small molecules (e.g., PROTACs, molecular glues), navigate to heliOS, create a New Assay Workflow, add Custom Assay, load Y-Structure FRET Kinetics, and modify parameters as needed.

Suggested assay parameters (e.g., flow rates, functionalization time, LED power) are available within the heliOS assay setup.

IMPORTANT: For binary interaction in red, set LED red ≥ 1. Remember to reset it to 0 when FRET interaction is under investigation. For more details, refer to the heliX+ guide.

Alternatively, the Y-Structure FRET Kinetics - auto LED assay can be used. This feature automatically adjusts LED power for optimal fluorescence signal and signal-to-noise ratio, which is highly recommended for weak binders or screening applications.

For studying bispecific antibodies with weak affinities (e.g., Hemlibra binding to Factor X and IX), navigate to heliOS, create a New Assay Workflow, add 'Simply Kinetics with Functionalization' from the Custom Assay list, and modify parameters as needed.

TIP: Antibodies are large proteins that may prevent dyes from being in close proximity, thus hindering FRET recording. Therefore, classic kinetics workflow and Fluorescence Proximity Sensing (FPS) are used for detecting binding in such cases.

For further questions, contact the support team at support.dbs@bruker.com.

Useful Order Numbers

Table 2. Order Numbers

Product Name Amount Order No
Y-structure Amine coupling kit 1 - Red 3 conjugations HK-NYS-NHS-1
Y-structure Amine coupling kit 2 - Green 3 conjugations HK-NYS-NHS-2
New Y-structure Kit 1: for proximity binding assay Spot 1 400 nM x 250 µL HK-NYS-1
New Y-structure His Capture Kit 500 nM x 200 µL HK-NYS-NTA

Contact

Dynamic Biosensors GmbH

Perchtinger Str. 8/10
81379 Munich
Germany

Bruker Scientific LLC

40 Manning Road, Manning Park
Billerica, MA 01821
USA

Order Information: order.biosensors@bruker.com

Technical Support: support.dbs@bruker.com

www.dynamic-biosensors.com

Instruments and chips are engineered and manufactured in Germany.

©2025 Dynamic Biosensors GmbH

For Research Use Only. Not for use in clinical diagnostic procedures.

Models: HK-NYS-2, HK-NYS-2 Maghreb Pharma Expo, HK-NYS-2 Maghreb Pharma Expo, Pharma Expo, Expo

File Info : application/pdf, 6 Pages, 1.85MB

PDF preview unavailable. Download the PDF instead.

HK-NYS-2

References

HexaPDF version 1.4.0

Related Documents

Preview Bruker Biosensors helix+ Y-Structure Kit 1: User Manual for Proximity Binding Assays
Comprehensive user manual for the Bruker Biosensors helix+ Y-Structure Kit 1 (HK-NYS-1). Learn about key features, contents, preparation steps, and assay setup for studying ternary complex formation and binary binding using FRET and Fluorescence Proximity Sensing (FPS).
Preview Bruker Biosensors heliX+ New Y-Structure His Capture Kit User Manual
Comprehensive user manual for the Bruker Biosensors heliX+ New Y-Structure His Capture Kit (HK-NYS-NTA), detailing key features, preparation, assay setup, and order information for histidine-tagged protein capture.
Preview Bruker Biosensors heliX+ HIS CAPTURE KIT 2 User Manual
User manual for the Bruker Biosensors heliX+ HIS CAPTURE KIT 2, detailing its features, adapter chip overview, product description, preparation steps, and useful order numbers for histidine-tagged protein capture.
Preview Bruker Bioscience Helix Cyto Normalization Solution (Red Dye) User Manual
User manual for the Bruker Bioscience Helix Cyto Normalization Solution (Red Dye), detailing its features, product description, preparation, and application notes for scIC measurements.
Preview Bruker Biosensors Amine Coupling Kit 2 User Manual
User manual for the Bruker Biosensors Amine Coupling Kit 2, detailing the process for coupling molecules with primary amines to the ligand strand using spin column purification.
Preview Bruker Biosensors heliX cyto Labeling Kit Green Dye User Manual
User manual for the Bruker Biosensors heliX cyto Labeling Kit Green Dye, detailing its features, a 3-step labeling workflow, product contents, required materials, troubleshooting, and order information for labeling protein analytes with a green fluorescent dye.