dynamic BIOSENSORS HK-MAL-2 Thiol Coupling Kit 2 User Manual

dynamic BIOSENSORS HK-MAL-2 Thiol Coupling Kit 2

Specifications:

• Product Name: heliX+ THIOL COUPLING KIT 2
• Order Number : HK-MAL-2
• Manufacturer : Dynamic Biosensors GmbH & Inc.

Product Description:
The heliX+ THIOL COUPLING KIT 2 is designed for coupling molecules with free thiol groups to the ligand strand using a spin column purification method. It includes all necessary materials for thiol coupling and purification, ensuring a streamlined workflow.

Workflow Overview:

1. DNA Modification: Functionalize the Ligand strand with free thiol reactive groups.
2. Ligand Conjugation: Add the biomolecule (ligand) to the functionalized Ligand strand and incubate.
3. Purification: Purify the Ligand strand conjugate using the provided anion exchange spin columns.
4. Ready-to-use: After buffer exchange, aliquot and store the conjugates.

Product Usage Instructions

1. DNA Modification:
   Functionalize the Ligand strand with free thiol-reactive groups as per the protocol provided.

2. Ligand Conjugation:
   Add the biomolecule (ligand) to the functionalized Ligand strand and incubate for at least 1 hour following the recommended conditions.

3. Purification:
   Purify the Ligand strand conjugate using the anion exchange spin columns provided. Perform buffer exchange as recommended before storing the conjugates.

4. Ready-to-use:
   After purification and storage, the Ligand strand conjugates are ready for further use in experiments or applications.

Key Features

• Allows for coupling of biomolecules with free thiols (e.g. cysteines) to the Ligand strand in a single reaction tube.
• Convenient standard chemistry (Maleimide chemistry).
• Compatible with heliX® Adapter Chip and multipurpose chips.
• Compatible with spin column purification of the ligand-DNA conjugate (> 7 kDa).
• Coupling of multiple ligands can be performed simultaneously.
• Yields > 95 % pure ligand-DNA conjugate with user determined quality of final product.
• Includes reagents for five individual conjugation reactions (approx. 10-50 regenerations each; up to max. 500).
• Compatible with automated standard regeneration process.

Workflow Overview

3-Step Conjugation Workflow

Timeline : Hands-on time < 1 h | Incubation ~ 2 h | Total ~ 3 h

Product Description

Order Number : HK-MAL-2

Table 1. Contents and Storage Information

For research use only.
This product has a limited shelf life, please see expiry date on label.

IMPORTANT: Products might be shipped at different temperature, but the storage should respect the indications reported in Table 1.

The kit contains reagents sufficient for 5 conjugations of approx. 50-200 µg biomolecule each. The resin slurry of the Purification spin column contains 0.02 % sodium azide.

Additional Materials Required

Table 2. Additional Materials

x g
Vortex|
1.5 mL reaction tubes|
UV-Vis Spectrophotometer (e.g. Nanodrop)| For determination of the Ligand strand conjugate’s concentration

All necessary solutions and buffers are included in the kit.

Important Notes

1. The lyophilized Ligand strand is not always found at the bottom of the tube, but may remain on the tube wall or in the tube cap. Please, always check the presence of the lyophilized Ligand strand, which has the appearance of a clear pellet (it may be necessary to remove the tube label to see it). If it is not on the bottom, please spin down (at high speed) the tube for couple of minutes before dissolving the DNA in buffer or, in alternative, place the tip of your pipette in proximity of the DNA pellet and dispense the buffer directly on it; the DNA will quickly dissolve.
2. The crosslinker will be linked to the free thiol groups of the ligand.
3. Do not use 2-Mercaptoethanol or other thiol-based reducing agents during conjugation process. If a reducing agent is necessary, TCEP is recommended up to 1 mM.
4. Avoid using partially purified protein samples or protein samples containing carriers (e.g. BSA).
5. To ensure the highest reaction yields, the ligand should be dissolved in Buffer A. Buffer exchange is recommended prior to the conjugation process.
6. Before starting, briefly centrifuge all tubes with blue, brown and transparent caps to ensure that all material is at the bottom of the tubes.
7. If the pI of the protein is < 6, a low pH buffer for conjugation (Order No: BU-M-150-1) is recommended. For more information, please email support@dynamic-biosensors.com.

3-Step Conjugation of a Biomolecule to a Ligand strand

Please read the entire protocol before starting and perform all steps without interruption. TIP This protocol can be performed simultaneously for multiple coupling reactions. Avoid using partially purified protein samples or protein samples containing carriers (e.g., BSA).

Before starting allow the crosslinker to reach room temperature before use.

Nanolever Modification

1. Dissolve Ligand strand MAL in 40 μL Buffer C prior to use, vortex until all solids are completely dissolved and briefly spin down.

2. Dissolve the crosslinker (green cap) by adding 100 μL ddH2O, vortex until all solids are completely dissolved and briefly spin down. IMPORTANT: Always use fresh compound.

3. Add 10 μL of the freshly prepared linker solution to one Ligand strand aliquot. Discard the remaining linker solution from step 2.

4. Vortex the reactants for 10 sec, spin down and incubate for 45 minutes at room temperature.
   IMPORTANT Do not exceed incubation time or the reaction yield will decrease.

5. In the meantime, equilibrate two purification spin columns (red cap) for one coupling reaction:

6. Remove the column’s bottom seal and loosen cap (do not remove cap).

7. Place the column in a 2.0 mL reaction tube.

8. Centrifuge at 1,500 × g for 1 minute to remove the storage solution.

9. Add 400 μL of Buffer A to the column’s resin bed. Centrifuge at 1,500 × g for 1 minute to remove buffer.

10. Repeat step d and discard the resulting buffer from the reaction tube. The purification spin column should now be in a dry state.

11. Sample loading

12. Place the columns from step 5 in new 1.5 mL reaction tubes.

13. Remove the cap of spin column number 1 and apply the sample from step 4 to the top of the resin bed.

14. Centrifuge at 1,500 × g for 2 minutes to collect the sample (flow-through). Discard the Purification spin column after use.

15. Remove the cap of spin column number 2 and apply the sample from step c to the resin bed.

16. Centrifuge at 1,500 × g for 2 minutes to collect the sample (flow-through). Discard the Purification spin column after use.

Ligand Conjugation

1. Add approx. 100 μg (up to a maximum of 200 μg) of the ligand (concentration approx. 0.5 – 50 mg/mL) to the sample from step 6. For optimal conditions use a volume of approx. 50 μL.
   EXAMPLE : Adjust protein concentration to 2 mg/mL and use 50 μL for conjugation.
   IMPORTANT Ensure the storage buffer of the ligand does not contain any thiols, e.g. 2-Mercaptoethanol (please check Important Notes).

2. Mix the reaction by pipetting up and down and let it react at room temperature for at least 1 hour.

IMPORTANT Do not vortex. If necessary, the reaction can be carried out at 4 °C with a longer reaction time (e.g. overnight).

Spin column Purification and Buffer Exchange

1. Perform a purification reaction using anion exchange spin columns.

2. To equilibrate the spin column add 400 μL Buffer A and put the spin column in the provided collection tube. Centrifuge at 2,000 x g for 5 minutes and discard the flow-through.
   Note : To achieve even liquid flow-through the membrane using a fixed- angle rotor, align the printed letter (Q) toward the center of the rotor for all chromatography steps.

3. Add the complete sample to the spin column, incubate for 1 minute and centrifuge for 2 minutes at 2,000 x g. Discard the flow-through.

4. Place the column in a new collection tube and add 100 μL of the Elution Buffer E48 to the spin column, incubate for 5 minutes and centrifuge for 2 minutes at 2,000 x g. Repeat the elution step and combine the two flow-throughs.

5. Add 200 μL of PE40 (or TE40, HE40) buffer to the eluted and combined sample from step c.

6. IMPORTANT If the protein is not stable in PE40 (or TE40, HE40), please check buffer compatibility with the switchSENSE® compatibility sheet.

7. Apply the sample to the centrifugal filter unit and centrifuge at 13,000 x g (up to 14,000 x g) for 10 minutes and discard flow-through. (Please check Additional information: Buffer Exchange and Concentration with Centrifugal Filter Units).

8. Add 350 μL of PE40 (or TE40, HE40) buffer and centrifuge at 13,000 x g for 10 minutes. Discard the flowthrough.

9. Add 350 μL of PE40 (or TE40, HE40) buffer and centrifuge at 13,000 x g for 15 minutes. Discard the flowthrough.

10. To recover the Ligand strand conjugate, place the centrifugal filter unit upside down in a new centrifugal collection tube (provided in the kit). Spin at 1,000 x g for 2 minutes to transfer the sample to the tube.

Aliquots and Storage

1. Measure the absorbance of the Ligand strand conjugate at 260 nm on a UV-Vis Spectrophotometer (e.g. Nanodrop).
2. Determine the concentration of the Ligand strand conjugate by inserting into the following equation: where d is the path length (usually equal to 1 cm; however, please check the UV-Vis Spectrophotometer user manual)
3. For a ready to use solution for a biochip functionalization, please adjust the concentration to 500 nM (or up to 1 μM) with PE40 (or TE40, HE40) buffer (including up to 10 % glycerol, if needed) and prepare 20 μL aliquots.
4. Store between -86 °C and 8 °C, as desired. Stability of the solution is related to the stability of the ligand molecule.

IMPORTANT Before a switchSENSE® interaction measurement, please add the appropriate adapter strand to the conjugate solution.

Additional Information

Buffer Exchange and Concentration with Centrifugal Filter Units

1. Take one centrifugal filter unit, add the appropriate volume of buffer in the filter device, and cap it.
2. Place capped filter device into the centrifuge rotor, aligning the cap strap toward the center of the rotor; counterbalance with a similar device.
3. Spin the device at 13,000 x g (or 14,000 x g) for the given time.
4. Remove the flow through and repeat steps 1-3.
5. Remove the assembled device from the centrifuge and separate the filter device from the microcentrifuge tube.
6. To recover the conjugate, place the filter device upside down in a clean centrifugal tube, aligning open cap towards the center of the rotor; counterbalance with a similar device. Spin for 2 minutes at 1,000 x g to transfer the sample from the device to the tube.

Compatibility Sheet

Buffer additives
The conjugation of ligands with all available coupling kits can be performed with many different additives. The following list shows all tested ones, but please note that others not listed here may also be successfully used.

1. thiol-based reducing agents
2. contains primary amines
3. caution, may harm the ligand

pH/pI
The pH value for the conjugation buffer may range from pH 5.0 to pH 8.0, depending on the ligand characteristics. When performing a conjugation of proteins with a pI of < 6, please note that using a buffer with lower pH may result in a better yield of conjugate.

Salt concentration
For standard conjugations 50 mM buffer salt and 150 mM NaCl (monovalent salt) are used. When performing conjugation of strongly charged ligands, make sure that the concentration of NaCl is sufficiently high (up to 400 mM NaCl is recommended). Otherwise, precipitation of DNA may occur. The shielding effect of monovalent sodium cations leads to DNA stabilization through the neutralization of the negative charge on the sugar-phosphate backbone.

Useful Order Numbers

Table 3. Order Numbers

HK-NHS-3
Centrifugal filter unit (3 kDa MWCO)| 5 pcs.| CF-003-5
Centrifugal filter unit (10 kDa MWCO)| 5 pcs.| CF-010-5
10x Buffer A [ 1]| 50 mL (yielding 500 mL)| BU-P-150-10
1x Buffer M [ 6]| 50 mL| BU-M-150-1

Contact

• Dynamic Biosensors GmbH Perchtinger Str. 8/10 81379 Munich Germany Dynamic Biosensors, Inc. 300 Trade Center, Suite 1400 Woburn, MA 01801 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.

©2024 Dynamic Biosensors GmbH | Dynamic Biosensors, Inc. All rights reserved

1. Buffer A: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.2
2. Buffer C: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 8.0
3. Buffer PE40: 10 mM Na2HPO4/NaH2PO4, 40 mM NaCl, pH 7.4, 0.05 % Tween, 50 μM EDTA, 50 μM EGTA
4. Buffer E48: 50 mM Na2HPO4/NaH2PO4, 618 mM NaCl, pH 7.2
5. For conjugation of proteins with a molecular weight higher than 20 kDa: Centrifugal filter units with a MWCO of 10 kDa can be ordered for a faster concentration process (Order No: CF-010-5).
6. Buffer M: 50 mM MES, 150 mM NaCl, pH 6.5

www.dynamic-biosensors.com

FAQ

Q: Can I use 2-mercaptoethanol during the conjugation process?
A: It is not recommended to use 2-Mercaptoethanol or other thiol-based reducing agents. If necessary, TCEP up to 1 mM is suggested.

Q: What should I do if my protein sample has a pI less than 6?
A: For proteins with a pI less than 6, it is recommended to use a low pH buffer for conjugation. Contact support@dynamic- biosensors.com for more information.

References

• HomePage | Biosensors International Ltd
• Home - Dynamic Biosensors
• Home - Dynamic Biosensors

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