Jun 05, 2026
Purification of Antibody–Oligo Conjugates Using the Conjugation CleanAb Kit (Agarose)
- Marina Able1
- 1Proteintech Germany GmbH
- Proteintech
Protocol Citation: Marina Able 2026. Purification of Antibody–Oligo Conjugates Using the Conjugation CleanAb Kit (Agarose). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7qr81lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: June 05, 2026
Last Modified: June 05, 2026
Protocol Integer ID: 318607
Keywords: Antibody-oligonucleotide conjugate, Oligonucleotide removal, Antibody-oligonucleotide purification, Antibody purification, DNA-barcoded antibodies, Antibody-oligonucleotide labeling, free oligo removal, ultrafiltration, amicon, conjugation cleanab kits agarose for rabbit igg, purification of antibody, conjugation cleanab kits agarose, antibody integrity, conjugation cleanab kit, antibody, damage to antibody integrity, labeled antibody, major bottleneck in antibody, incomplete removal of free oligonucleotide, coated agarose bead, purification, easy removal of excess oligo, proteintech, oligo conjugation, conjugated rabbit, bead conjugation, oligo conjugate, agarose, free oligonucleotide, rabbit igg, conjugate, extension assay, mouse igg
Abstract
High-quality oligo-labeled antibodies are crucial for barcoded imaging applications, proximity ligation and extension assays, CITE-seq, and more.
A major bottleneck in antibody–oligo conjugation is the purification of antibody–oligonucleotide conjugates, which often suffers from low recovery, incomplete removal of free oligonucleotides, and damage to antibody integrity, ultimately resulting in inconsistent results.
Conjugation CleanAb Kits Agarose for Rabbit IgG (Proteintech, CK001) or Mouse IgG (Proteintech, CK003) are designed for fast & easy removal of excess oligos from oligo-conjugated rabbit antibodies post conjugation or after on-bead conjugation. The Kits use coated agarose beads optimized for elution at native pH (pH 7.5), facilitating gentle recovery of your sample from the beads, with high efficiency and excellent recovery rates.
General Notes
Conjugation CleanAb Kits facilitate fast & easy clean-up of conjugated rabbit IgG (Proteintech, Cat
no. CK001) or mouse IgG (Proteintech, Cat no. CK003) to remove excess oligonucleotide at near
native pH (pH 7.5) and with a high recovery rate.
- Conjugation CleanAb Kits are compatible with antibodies conjugated via common techniques such as NHS ester – amine reaction, maleimide – thiol reaction, or click-chemistry.
- All components needed for the protocol are supplied with the kit, except for Eppendorf tubes.
- 100 µl Beads (slurry) can be used for purification of up to 150 µg of conjugated primary antibody. Bead volumes can be adjusted proportionally to fit your experimental needs.
- The following protocol describes the use of 100 µl of CleanAb Beads (slurry).
There are two ways to use CleanAb:
Post conjugation clean-up (Option 1): Conjugate the IgG in solution using your preferred technique and use the CleanAb kit to remove free oligos afterwards.
On-bead conjugation and clean-up (Option 2): Immobilize the IgG on CleanAb beads, wash away any undesired buffer components, and perform your conjugation on the beads. This method removes the need for buffer exchange prior to conjugation to remove incompatible components (e.g., Tris, BSA).
Protocol for post conjugation clean-up (Option 1)
Equilibration of beads
Resuspend the beads by gently pipetting them up and down or by inverting the
tube. Do not vortex.
Remove stopper from a spin column (provided with the kit) and insert it into a 2 ml Eppendorf tube.
Transfer 100 µl beads (slurry) to a spin column.
Add 500 µl Wash buffer to the beads and centrifuge at 2000x g for 1 min. Discard the flow-through.
Binding of IgG-oligonucleotide
Close the spin column and add your conjugated primary rabbit or mouse IgG (up to 150 µg) to the equilibrated beads. Add wash buffer to a final volume of 100 µl if input volume is below that.
Incubate for 30 min at room temperature under end-over-end rotation.
Washing of beads
Open spin column and insert into a 2 ml Eppendorf tube.
Centrifuge the spin column at 2000x g for 1 min. Discard flow-through.
Resuspend beads in 500 µl Wash buffer.
Centrifuge the spin column at 2000x g for 1 min. Discard flow-through.
Repeat steps 10–11 twice.
Elution of IgG-oligonucleotide
Close spin column and add 100 µl Elution buffer (pre-equilibrated to room temperature) to beads.
Incubate beads at room temperature for 10 min under end-over-end rotation.
Open spin column and transfer to fresh 2 ml Eppendorf tube.
Centrifuge the spin column at 2000x g for 2 min.
Save the eluate, which will contain the purified IgG-oligonucleotide conjugate.
Repeat steps 13–16.
Pool eluates.
Optional: Buffer exchange to remove Elution buffer
Use a Zeba spin desalting column or similar and follow the manufacturer's instructions and use your desired buffer for long term storage.
Note: The Elution buffer contains high concentrations of certain reagents that might affect running behavior in SDS PAGE and concentration determination. If you need to measure concentrations precisely, we recommend doing the buffer exchange step.
For long-term storage at -20°C (> 6 months), buffer exchange is also recommended. Storage at -80°C does not require buffer exchange.
Please note that a buffer exchange can lead to minor sample loss.
On-bead conjugation and clean-up (Option 2)
Equilibration of beads
Resuspend the beads by gently pipetting them up and down or by inverting the
tube. Do not vortex.
Remove stopper from a spin column (provided with the kit) and insert it into a 2
ml Eppendorf tube.
Transfer 100 µl beads (slurry) to a spin column.
Add 500 µl Wash buffer to the beads and centrifuge at 2000x g for 1 min. Discard the flow-through.
Binding of IgG-oligonucleotide
Close the spin column and add your primary rabbit or mouse IgG (up to 150 µg) to the equilibrated beads. Add wash buffer to a final volume of 100 µl if input volume is below that.
Incubate for 30 min at room temperature under end-over-end rotation.
Washing of beads
Open spin column and insert into a 2 ml Eppendorf tube.
Centrifuge the spin column at 2000x g for 1 min. Discard flow-through.
Resuspend beads in 500 µL of a buffer appropriate for your conjugation technique.
Centrifuge the spin column at 2000x g for 1 min. Discard flow-through.
Repeat steps 27–28 twice.
On-bead conjugation
Close the spin column. Perform on-bead conjugation using the appropriate buffer and reagents for
your conjugation technique of choice. Use similar amounts as you would have
used for a conjugation in solution.
Note: If you use a two-step conjugation strategy (e.g., introduction of a click
chemistry handle in the first step, then the addition of the oligonucleotide using
click-chemistry in the second step), return to step 25. Otherwise, carry on with the next step.
Open spin column and insert into a 2 ml Eppendorf tube.
Centrifuge the spin column at 2000x g for 1 min. Discard flow-through.
Resuspend beads in 500 µL Wash buffer.
Centrifuge the spin column at 2000x g for 1 min. Discard flow-through.
Repeat steps 34–35 twice.
Elution of IgG-oligonucleotide
Close spin column and add 100 µl Elution buffer (pre-equilibrated to room temperature) to beads.
Incubate beads at room temperature for 10 min under end-over-end rotation.
Open spin column and transfer to fresh 2 ml Eppendorf tube.
Centrifuge the spin column at 2000x g for 2 min.
Save the eluate, which will contain the purified IgG-oligonucleotide conjugate.
Repeat steps 37–40.
Pool eluates.
Optional step: Buffer exchange to remove Elution buffer
Use a Zeba spin desalting column or similar and follow the manufacturer's
instructions and use your desired buffer for long term storage.
Note: The Elution buffer contains high concentrations of certain reagents that
might affect running behavior in SDS PAGE and concentration determination. If
you need to measure concentrations precisely, we recommend doing the buffer
exchange step.
For long-term storage at -20°C (> 6 months), buffer exchange is also
recommended. Storage at -80°C does not require buffer exchange.
Please note that a buffer exchange can lead to minor sample loss.
Protocol references