dynamic BIOSENSORS PF-AB-1 Antibody Oligo Conjugation Kit User Guide

dynamic BIOSENSORS PF-AB-1 Antibody Oligo Conjugation Kit

Product Information

• Specifications:
  • Product Name: proFIRE Antibody Oligo Conjugation Kit
  • Order Number: PF-AB-1
  • Amount: Various options available, refer to product description
  • For Use: Research purposes only
• Product Description:
  • The proFIRE Antibody Oligo Conjugation Kit allows for the functionalization of DNA via amines (-NH2). It includes all necessary solutions and buffers for the conjugation process.
• Workflow Overview:
  • The 3-step conjugation workflow involves DNA modification, protein conjugation, and purification. The process results in ready-to-use fractions of antibody-DNA conjugate.
• DNA Modification:
  • The DNA is activated with amine-reactive groups.
• Protein Conjugation:
  • The antibody is added to the functionalized DNA and incubated for at least 1 hour.
• Purification:
  • The excess linker is removed by the spin column after incubation.
• Timeline:
  • Hands-on time: Less than 1 hour
  • Incubation: Approximately 2 hours
  • Total: Approximately 3 hours

Product Usage Instructions

• Additional Materials Required:
  • Oligo with DBCO modification
  • Benchtop microcentrifuge
  • Vortex
  • 1.5 mL reaction tubes
  • UV-Vis Spectrophotometer (e.g., Nanodrop)
• Important Notes:
  • Avoid using buffers containing primary amines during the conjugation process.
  • Dithiothreitol (DTT) can be used up to 1 mM during the conjugation process.
  • Avoid using partially purified protein samples or those containing carriers.
  • Ligand should be dissolved in Antibody Buffer for optimal reaction yields.
• Conjugation Process:
  • Follow the 3-step conjugation process as outlined in the manual. Ensure to use the control oligo provided for confirmation of optimal chemistry.

FAQs

• Q: Can I use buffers containing primary amines during the conjugation process?
  • A: No, it is advised to avoid buffers containing primary amines to prevent interference with the conjugation process.
• Q: What is the incubation time for protein conjugation?
  • A: The antibody should be incubated with the functionalized DNA for at least 1 hour.

Key Features

• Allows for coupling of antibodies with primary amines (e.g. NH2-terminus, lysines) to modify DNA in a reaction tube.
• Sufficient for 3 reactions plus one control oligo.
• Convenient standard chemistry.
• Suitable for any antibody format.
• 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 antibodies 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| After incubation the excess| The antibody-DNA| The fractions with
amine-reactive groups.| the linker is removed by spin| conjugate is purified using| antibody-DNA conjugate
 | column. The antibody is| the pro FIRE® system.| are ready for further
 | added to the functionalized|  | processing.
 | DNA and incubated for at|  |
 | At least 1 h.|  |

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

Product Description

• Order Number: PF-AB-1

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 may be shipped at different temperatures, but storage should adhere to the guidelines outlined in the Table.
• The kit contains reagents sufficient for three conjugations of approximately 50-500 μg of antibody each.
• The resin slurry in the purification spin column contains 0.02 % sodium azide.

Additional Materials Required

Table 2. Additional Materials

with a Thiol, reduced, HPLC 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 Antibody 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.

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).

IMPORTANT

• Each conjugation kit is supplied with a control oligo (a 48 base oligo with a 5’-DBCO).
• This oligo is included as positive control in order to give the option of confirming the conjugation chemistry is working optimally.
• Please proceed with the control oligo as with your oligo of choice.

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

1. DNA Modification
2. Dissolve the DNA in 40 μL Antibody Buffer prior to use, vortex until all solids are completely dissolved and briefly spin down.
3. Dissolve the crosslinker (brown cap) by adding 100 μL ddH2O, 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 2.
5. Vortex the reactants for 10 sec, spin down and incubate for 20 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:
  *  **a.** Remove the column’s bottom seal and loosen cap (do not remove 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.
6.  **Sample loading**
  *  **a.** Place the columns from step 5 in new 1.5 mL reaction tubes.
  *  **b.** Remove the cap of spin column number 1 and apply the sample from step 4 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. Antibody Conjugation
3. Add approx. 100 μg (up to a maximum of 500 μg) of the antibody (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 antibody concentration to 5 mg/mL and use 20 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 Antibody Buffer.
   • b. If the volume exceeds 160 μL, please perform 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. Example chromatograms are shown in the Additional Information section: proFIRE® purification of an antibody-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 5 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 5 minutes. Discard the flowthrough.
8. Add 350 μL of buffer of choice for buffer exchange and centrifuge at 13,000 x g for 10 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 antibody-DNA conjugate at 260 nm (= ) on a UV-Vis Spectrophotometer (e.g. Nanodrop).
12. Determine the concentration of the antibody-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 antibody.

Additional Information proFIRE® purification of an antibody-DNA conjugate

1. We recommend using the proFIRE® system equipped with an ion exchange column, Buffer A [1] and Buffer B [2], which have the same composition, but different salt concentrations, allowing peak separation. In Figure 1 a typical proFIRE® chromatogram of a 100 μg antibody-DNA conjugate purification is depicted, while in Figure 2 a typical proFIRE® chromatogram of a 500 μg antibody-DNA conjugate purification is depicted. In both cases, the peak of the antibody-DNA conjugate is separated from the free antibody (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 (e.g., in Figure 1: fractions 6-7 and 9-10), 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 a 100 μg antibody-DNA conjugate purification. Used buffers: Buffer A [1]; Buffer B [2]. Column: DBS-chromatographic column. Flow: 1 mL/min. Used program: DNA length 48, Type 1.
   • Figure 2. proFIRE® chromatogram a 500 μg antibody-DNA conjugate purification. Used buffers: Buffer A [1]; Buffer B [2]. Column: DBS-chromatographic column. Flow: 1 mL/min. Used program: DNA length 48, Type 1.
   • NOTE The crosslinker will be linked to the amino groups (NH2-groups) of antibodies. Among the four amino acids with side-chain amino groups (glutamine, lysine, arginine, asparagine), only lysine can react. In addition, the N-terminal amino group can be functionalized with the oligo, too. Thus, many sites can be linked via the crosslinker as shown in the diagram below.

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 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 the 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
pro FIRE® Thiol Coupling Kit 1 for proteins (>5 kDa);| 5 conjugations| PF-SH-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 [ 1 ]| 50 mL (yielding 500 mL)| PF-BU-A-10
5x Buffer B [ 2 ]| 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. Buffer A: 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.2
2. Buffer B: 50 mM Na2HPO4/NaH2PO4, 1 M NaCl, pH 7.2

www.dynamic-biosensors.com.

Antibody Oligo Conjugation Kit

• Functionalization of DNA via amines (-NH2)
• Dynamic Biosensors GmbH & Inc. PF-AB-1 v7.1

References

• HomePage | Biosensors International Ltd
• Home - Dynamic Biosensors
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