Bruker Biosensors heliX+ New Y-Structure His Capture Kit User Manual

Key Features

Designed for capture of histidine-tagged proteins (His6 or His10) using Tris-NTA.
Includes Ligand strand 1 - Red and Ligand strand 2 - Green with Tris-NTA for 20 regenerations each.
Specifically for the new Y-structure kit.
Facilitates dimerization projects.
Enables easy coupling of homo-/hetero-proteins to the arms via his-tag capture.
Compatible with heliX® Adapter Chip, both Spot 1 and Spot 2.

heliX® Adapter Chip Overview

The heliX® Adapter Chip features 2 spots with 2 different anchor sequences for DNA-encoded addressing. Spot 1 is functionalized with the capture molecule, while Spot 2 is used as a real-time reference.

Single Capture

Illustrates a single capture setup on the heliX® Adapter Chip. Spot 1 shows 'Anchor strand 1' functionalized with 'Ligand strand 1 - Red - NTA' and a red dye. Spot 2 shows 'c-Anchor strand 2' functionalized with 'Ligand strand 2 - Green - NTA' and a green dye, serving as a reference.

Double Capture

Depicts a double capture scenario on the heliX® Adapter Chip, similar to single capture but showing captured histidine-tagged proteins on both the red (Spot 1) and green (Spot 2) ligand arms.

Product Description

Order Number: HK-NYS-NTA

Table 1. Contents and Storage Information

Material	Cap	Concentration	Amount	Buffer	Storage
New Y-structure Red Adapter strand - NTA	Purple	500 nM	2 x 100 µL	TE40 [1]	-20°C
New Y-structure Green Adapter strand - NTA	Purple	500 nM	2 x 100 µL	TE40 [1]	-20°C
Loading Solution (NiCl₂)	Transparent	10 mM	10 x 1500 µL	TE40 [1]	-20°C
Imidazole Solution	Transparent	250 mM	10 x 2000 µL	TE140 [2]	-20°C

For research use only. This product has a limited shelf life, please see expiry date on label. After preparation of ready-to-use solution, the expiry date is 6 months.

Preparation

Step 1

For surface functionalization, the Y-structure Red Adapter strand (harboring the red dye Ra) and the Y-structure Green Adapter strand (harboring the green dye Ga) need to be pre-hybridized with either the new Y-structure Red Adapter strand - NTA or new Y-structure Green Adapter strand - NTA and a conjugated Ligand strand.

Example: In-solution hybridization of Y-structure strands with a combination of covalent coupling protein in green & his-tag capture strategy in red:

New Y-structure Red Adapter strand with Ra (400 nM)
New Y-structure Green Adapter strand with Ga (400 nM)
New Y-structure Red Adapter strand - NTA (500 nM)
Covalently conjugated protein to Y-structure Ligand strand 2 - Green (500 nM)

Combine at a 1:1 ratio (v/v).

Incubate the solution of step i) at room temperature (RT) for at least 2 hours to ensure complete hybridization. Overnight incubation at 4°C is also possible.

Step 2

Mix solution of step ii) and cAnchor strand 2 (100 nM) at 1:1 ratio (v/v).

Step 3

Solution is ready to use for heliX® Adapter Chip functionalization.

Example Volumes

Required volume for one functionalization for a combination of amine coupling and capture on the new Y-structure: 35 µL with a final concentration of 50 nM.

	Vial 1	Vial 2
New Y-structure Red Adapter strand with Ra (400 nM)	4.5 µL
New Y-structure Green Adapter strand with Ga (400 nM)	4.5 µL
Y-structure Red Adapter strand - NTA (500 nM)	4.5 µL
Conjugated protein to Y-structure Ligand strand 2 (500 nM)	4.5 µL
cAnchor strand 2 (100 nM)		18 µL

Assay Setup in heliOS

This specific kit requires a customized method consisting in His-tag capture plus Y-Structure FRET Kinetics, which is currently not provided among the verified assays. It can be easily created by an advanced heliOS user by applying the default parameters already existing in the two different and separate workflows (please refer to the heliX+ guide available at this link). For assistance in creating the new method, please contact the support team at support@dynamic-biosensors.com.

TIP: As the stability of his capture is affected by the protein, in case of long dissociations, consider using the classic conjugation approach.

Useful Order Numbers

Product Name	Comment	Order No
heliX® Adapter Chip	Chip with 2 detection spots	ADP-48-2-0
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 Kit 2: for proximity binding assay Spot 2	400 nM x 250 µL	HK-NYS-2

Contact Information

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@dynamic-biosensors.com

Technical Support: support@dynamic-biosensors.com

www.dynamic-biosensors.com

Instruments and chips are engineered and manufactured in Germany.

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

Buffer Definitions

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

[2] TE140: 10 mM Tris, 40 mM NaCl, 0.05 % Tween20, 50 µM EDTA, 50 µM EGTA

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Bruker Biosensors heliX+ User Manual

NEW Y-STRUCTURE HIS CAPTURE KIT