dynamic BIOSENSORS PF-NH2-1 Amine Coupling Kit User Guide

dynamic BIOSENSORS PF-NH2-1 Amine Coupling Kit

Product Information

• Specifications
  • Product Name: proFIRE Amine Coupling Kit 1 for Proteins (> 5 kDa)
  • Model: PF-NH2-1 v6.1
  • Order Number: PF-NH2-1
• Product Description
  • The profile Amine Coupling Kit 1 is designed for the functionalization of thiol-DNA via amines (-NH2) for proteins with a molecular weight greater than 5 kDa.
  • It includes all necessary materials and buffers for a 3-step conjugation workflow to efficiently link proteins to DNA.
• Key Features
  • 3-step conjugation workflow
  • Includes all required materials and buffers
  • Hands-on time less than 1 hour
  • Total processing time approximately 3 hours
• Workflow Overview
  • DNA Modification: Activate DNA with amine-reactive groups.
  • Protein Conjugation: Add protein/peptide to functionalized DNA and incubate.
  • Purification: Remove excess linker using a spin column.

Specifications and Contents

Product Usage Instructions

• Preparation
  • Before starting, ensure all materials are at the bottom of the tubes by centrifuging tubes with green and transparent caps briefly.
• Conjugation Process
  • Dissolve the ligand in the Conjugation Buffer.
  • Avoid buffers with primary amines during the process.
  • Activate DNA with amine reactive groups.
• Purification
  • After incubation, remove the excess linker using the provided spin column according to the kit instructions.
• Finalization
  • The fractions containing the protein-DNA conjugate are now ready for further processing or analysis.

FAQs

• Q: Can I use other reducing agents during the conjugation process?
  • A: Avoid using thiol-based reducing agents other than up to 1 mM of Dithiothreitol (DTT). If necessary, TCEP is recommended.
• Q: What should I do if my protein has a pI less than 6?
  • A: A lower pH buffer is necessary for proteins with a pI less than 6. Contact support@dynamicbiosensors.com. for more information.

Key Features

• Allows for coupling of biomolecules with primary amines (e.g. NH2-terminus, lysines) to thiol-modified DNA in a reaction tube.
• Oligos are not included in the kit.
• Convenient standard chemistry.
• Suitable for proteins and peptides (MW > 5 kDa).
• Suitable for any DNA sequence and length up to 150 bases.
• Yields >95 % pure protein-DNA conjugate with controlled quality of your product.
• Coupling of multiple proteins can be performed simultaneously.

Workflow Overview

3-Step Conjugation Workflow

1. DNA Modification| 2. Protein Conjugation| 3. Purification| 4. Ready-to- use
---|---|---|---
The DNA is activated with amine-reactive groups.| After incubation, the excess linker is removed by the spin column. The protein/peptide is added to the functionalized DNA and incubated for at least 1 h.| The protein-DNA conjugate is purified using the pro FIRE® system. After buffer exchange, the conjugates are aliquoted and stored.| The fractions with protein- DNA conjugate are ready for further processing.

Timeline:

• Hands-on time < 1 h
• Incubation ~ 2 h
• Total ~ 3 h

Product Description

• Order Number: PF-NH2-1

Table 1. Contents and Storage Information

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

IMPORTANT

• Products may be shipped at different temperatures, but storage should adhere to the guidelines outlined in the Table.
• The kit contains reagents sufficient for five conjugations of approximately 50-200 μg of biomolecule each.
• The resin slurry in the purification spin column contains 0.02 % sodium azide.

Additional Materials Required

Table 2. Additional Materials

grade) for one reaction
Benchtop microcentrifuge| Required speed range of between 1,000 x g to 13,000 x g
Vortex|
1.5 mL reaction tubes|
UV-Vis Spectrophotometer (e.g. Nanodrop)| For determination of the protein-DNA conjugate’s concentration

All necessary solutions and buffers are included in the kit.

Important Notes

• a. Avoid using any buffers containing primary amines (i.e. Tris, Glycine) during the conjugation process (Please check the Compatibility Sheet section).
• b. Up to 1 mM of Dithiothreitol (DTT) can be used during the conjugation process. Avoid using 2-Mercaptoethanol or any other thiol-based reducing agents during the conjugation process. If a reducing agent is necessary, TCEP is recommended up to 1 mM.
• c. Avoid using partially purified protein samples or protein samples containing carriers (e.g. BSA).
• d. To ensure the highest reaction yields, the ligand should be dissolved in a Conjugation Buffer. Buffer exchange is recommended before the conjugation process.
• e. Before starting, briefly centrifuge all tubes with green and transparent caps to ensure that all material is at the bottom of the tubes.
• f. For molecules with a molecular weight around or lower than 5 kDa, extra caution is required during the purification process. Small molecules and some peptides may not be properly purified using the provided chromatographic column. For more information please email support@dynamic-biosensors.com.
• g. If the pI of the protein is < 6, a lower pH buffer is necessary. For more information, please email support@dynamicbiosensors.com.

3-Step Conjugation of a Biomolecule to DNA in a Reaction Tube

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.

1. DNA Modification
2. Equilibrate two purification spin columns (red cap) for one coupling reaction:

  *  **a.** Remove the column’s bottom seal and loosen the cap (do not remove the cap).
  *  **b.** Place the column in a 2.0 mL reaction tube.
  *  **c.** Centrifuge at 1,500 × g for 1 minute to remove the storage solution.
  *  **d.** Add 400 μL of Conjugation Buffer to the column’s resin bed. Centrifuge at 1,500 × g for 1 minute to remove buffer.
  *  **e.** Repeat step d and discard the resulting buffer from the reaction tube. The purification spin column should now be in a dry state.
2. Dissolve the DNA in 40 μL Dilution Buffer before use, vortex until all solids are completely dissolved and briefly spin down.
3. Dissolve the crosslinker (green cap) by adding 100 μL ddH2O, and vortex until solids are completely dissolved and briefly spin down. IMPORTANT: Always use fresh compound.
4. Add 10 μL of the freshly prepared linker solution to one DNA aliquot. Discard the remaining linker solution from step 3.
5. Vortex the reactants for 10 sec, spin down, and incubate for 5 minutes at room temperature.
  *  **IMPORTANT** Do not exceed incubation time or the reaction yield will decrease.
6.  **Sample loading**
  *  **a.** Place the columns from step 1 in new 1.5 mL reaction tubes.
  *  **b.** Remove the cap of spin column number 1 and apply the sample from step 5 to the top of the resin bed.
  *  **c.** Centrifuge at 1,500 × g for 2 minutes to collect the sample (flow-through). Discard the Purification spin column after use.
  *  **d.** Remove the cap of spin column number 2 and apply the sample from step c to the resin bed.
  *  **e.** Centrifuge at 1,500 × g for 2 minutes to collect the sample (flow-through). Discard the Purification spin column after use.

2. Protein Conjugation
3. Add approx. 100 μg (up to a maximum of 200 μg) of the protein (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 primary amines, e.g. Tris buffers, or glycine (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).

3. proFIRE® Purification
   • Please refer to the proFIRE® User Manual.
   1. Perform a purification using the appropriate proFIRE® workflow (please refer to the proFIRE® User Manual). Please make sure that the sample volume is 160 μL.
   • a. If the volume is less than 160 μL, fill the missing volume with Conjugation Buffer.
   • b. If the volume exceeds 160 μL, please perform an additional 160 μL runs until all the sample is consumed.
   2. Use the Data Viewer software of the proFIRE® to identify which fractions contain pure conjugate.
   • An example chromatogram is shown in the Additional Information section: proFIRE® purification of a protein-DNA conjugate.
   3. Remove the recommended fractions from the fraction collector.
4. Buffer Exchange
5. Add 500 μL of the first proFIRE® fraction containing the protein-DNA conjugate to the centrifugal filter unit. Centrifuge at 13,000 x g (up to 14,000 x g) for 10 minutes and discard flow-through.
6. Add the remaining fractions to the same filter unit and repeat the centrifugation step to collect all samples in one tube. (Please check Additional information: Buffer Exchange and Concentration with Centrifugal Filter Units).
7. Add 350 μL of buffer of choice for buffer exchange and centrifuge at 13,000 x g for 10 minutes. Discard the flowthrough.
8. Add 350 μL of buffer of choice for buffer exchange and centrifuge at 13,000 x g for 15 minutes. Discard the flowthrough.
9. To recover the protein-DNA 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.
10. Aliquots and Storage
11. Measure the absorbance of the protein-DNA conjugate at 260 nm (= ) on a UV-Vis Spectrophotometer (e.g. Nanodrop).
12. Determine the concentration of the protein-DNA conjugate ( ) by inserting ( ) in the following equation:

  *  **with**
    * : Concentration of the ligand strand
    * : Absorbance at 260 nm
    * : Extinction coefficient of the DNA at 260 nm
    * : Lightpath length (typically 1 cm; please check your UV-Vis Spectrophotometer’s user manual)
3. Store between -86 °C and 8 °C, as desired. The stability of the solution is related to the stability of the protein.

Additional Information

proFIRE® purification of a protein-DNA conjugate

1. We recommend using the proFIRE® system equipped with an ion exchange column, Buffer A [2] and Buffer B [3], which have the same composition, but different salt concentrations, allowing peak separation. In Figure 1 a typical proFIRE® chromatogram of a protein-DNA conjugate purification is depicted, where the peak of the protein-DNA conjugate is separated from the free protein (left) and the free DNA (right).
   • IMPORTANT: The proFIRE® system owns tailored software for the automatic recognition and quantitation of DNA conjugates.
2. After purification, collect the protein-DNA conjugate fractions (Figure 1: fractions 8-10), concentrate the conjugate, and exchange buffer with your buffer of choice using a Centrifugal filter unit, as described in section II.
   • Figure 1. proFIRE® chromatogram of a ligand strand conjugate purification.
   • Used buffers: Buffer A [2]; Buffer B [3].
   • Column: DBS-chromatographic column.
   • Flow: 1 mL/min.
   • Used program: DNA length 48, Type 1.

Buffer Exchange and Concentration with Centrifugal Filter Units

1. Take one centrifugal filter unit, add the appropriate volume of buffer to 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 the 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 additives. Please note that other additives, which are not listed here may successfully be used for conjugation.

• thiol-based reducing agents
• contain primary amines
• caution, and 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 a 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 proteins, 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

PF-NH2-1
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 [ 2 ]| 50 mL (yielding 500 mL)| PF-BU-A-10
5x Buffer B [ 3 ]| 50 mL (yielding 250 mL)| PF-BU-B-5
Conjugation Buffer| 12 mL| PF-BU-C-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. 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).
2. Buffer A: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.2
3. Buffer B: 50 mM Na2HPO4/NaH2PO4, 1 M NaCl, pH 7.2

www.dynamic-biosensors.com.

Amine Coupling Kit 1 for Proteins ( > 5 kDa)

• functionalization of thiol-DNA via amines (-NH2)
• Dynamic Biosensors GmbH & Inc.
• PF-NH2-1 v6.1

References

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

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