Conjugation CleanAb Kits
Fast and efficient antibody clean-up of conjugated antibodies
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Compatible with Rabbit IgG or Mouse IgG (all subtypes)
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Native elution at pH 7.5
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High recovery rate of 60% - 90%
What are Proteintech Conjugation CleanAb Kits?
Conjugation CleanAb Kits are designed for fast & easy removal of excess oligo or unreacted dyes from conjugated primary antibodies - post conjugation or after on-bead conjugation.
The Kit uses coated agarose or magnetic 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.
CleanAb Kits are compatible with Rabbit IgG or Mouse IgG (all subtypes).
Kit components
- CleanAb Beads for Rabbit or Mouse IgG
- CleanAb Elution Buffer
- CleanAb Wash Buffer
- Spin columns
Immunostaining of HeLa cells with mouse anti-B23 antibodies (60096-1-Ig, 1:100, orange), on-bead chemically conjugated to a 15-nt ssDNA oligo, purified using CleanAB beads and desalted. Detection was performed by hybridization with an imaging DNA strand labeled with ATTO643.
Available CleanAb Kits
| Product | Available Sizes | Format | Application |
|---|---|---|---|
| CleanAb Kit for conjugated Rabbit IgG | 1 ml | Agarose, Magnetic Agarose | Oligo removal, dye removal |
| CleanAb Kit for conjugated Mouse IgG | 1 ml | Agarose, Magnetic Agarose | Oligo removal, dye removal |
Kit Compatibility
Oligo Removal: CleanAb kits have been validated for efficient removal of unconjugated oligonucleotides from oligo-conjugated antibodies across a wide range of commonly used oligo lengths (15-90 nt) and types, including oligos with Poly(A)-tail or phosphorothioate-modification, as well as for different types of end applications like CODEX, PLA/PEA, NULISA, or CITE-Seq.
Dye Removal: CleanAb kits have been validated for the efficient removal of excess fluorophores from conjugated antibodies across a wide range of dyes, including xanthene dyes (FITC), cyanine dyes (Cy3 and Cy5), Alexa Fluor™ dyes (Alexa Fluor™ 647 and 750), Atto dyes (Atto 647N), and Aquora™ dyes (Aquora™ 488, 550, and 650). The cleaned up antibody conjugates have been validated in IF imaging. The kit is not suitable for removal of PE or APC.
Kits are available in agarose and magnetic agarose format for clean-up of rabbit or mouse IgG. Refer to the respective product pages to access the protocols.
1 Kit, 2 Options - The CleanAb protocol at a glance
CleanAb Kits can be used in two ways: The clean-up can be performed after conjugation or the conjugation can be performed directly on the beads. The illustration below shows both approaches using oligonucleotide conjugation as an example. The same workflows also apply to dye removal.
IF of CD147 – Detection was performed by hybridization with an imaging DNA strand labeled with ATTO643.
Mouse IgG (anti-Lamin B1 IgG1; 66095-1-Ig) was conjugated with NHS-Azide (lane 1) and then with DBCO-Oligo (lane 2). (left) Post conjugation clean-up was done using CleanAb Kit for mouse IgG (lanes 3-5). Excess oligo was successfully removed as shown by SYBR Gold DNA staining and IgG eluted with high recovery (~70%) at neutral pH (lane 4). Desalting was done with Zeba spin desalting columns (lane 5). Only very little IgG remaining on beads (lane 6). (right) Post conjugation clean-up was done using Protein A beads (lanes 3-5). Excess oligo was successfully removed as shown by SYBR Gold DNA staining but IgG elution with low pH failed (lane 4). Most IgG remains on beads (lane 6)
Rabbit IgG (anti beta-tubulin; 80713-1-RR) was conjugated with NHS-Azide (lane 1) and then with DBCO-Oligo (lane 2). (left) Post conjugation clean-up was done using CleanAb Kit for rabbit IgG (lanes 3-5). Excess oligo was successfully removed as shown by SYBR Gold DNA staining and IgG eluted with high recovery (~70%) at neutral pH (lane 4). Desalting was done with Zeba spin desalting columns (lane 5). Only very little IgG remaining on beads (lane 6). (right) Post conjugation clean-up was done using Protein A beads (lanes 3-5). Excess oligo was successfully removed as shown by SYBR Gold DNA staining but IgG elution with low pH failed (lane 4). Most IgG remains on beads (lane 6)
(left) Mouse IgG (anti-Lamin B1 IgG1; 66095-1-Ig, lane 1) was bound to CleanAb rabbit beads (flowthrough in lane 2). then functionalized on beads with NHS-Azide (flowthrough in lane 3) and then with DBCO-Oligo (flowthrough in lane 4). Excess oligo was successfully removed as shown in SYBR Gold DNA staining and the oligo-conjugated IgG was eluted with high recovery at neutral pH (lane 6). Desalting was done with Zeba spin desalting columns (lane 8). Only very little IgG remaining on beads (lane 9). Note: the CleanAb Elution Buffer can interfere with SDS gels, which can make the CleanAb elution appear washed out. (right) Mouse IgG (anti-Lamin B1 IgG1; 66095-1-Ig, lane 1) was bound to Protein A beads (flowthrough in lane 2). Excess oligo was successfully removed as shown in SYBR Gold DNA staining but the oligo-conjugated IgG failed to elute at low pH (lane 6). Most IgG remains on beads (lane 9).
(left) Rabbit IgG (Isotype control, lane 1) was bound to CleanAb rabbit beads (flowthrough in lane 2). then functionalized on beads with NHS-Azide (flowthrough in lane 3) and then with DBCO-Oligo (flowthrough in lane 4). Excess oligo was successfully removed as shown in SYBR Gold DNA staining and the oligo-conjugated IgG was eluted with high recovery at neutral pH (lane 6). Desalting was done with Zeba spin desalting columns (lane 8). Only very little IgG remaining on beads (lane 9). Note: the CleanAb Elution Buffer can interfere with SDS gels, which can make the CleanAb elution appear washed out. (right) Rabbit IgG (Isotype control, lane 1) was bound to Protein A beads (flowthrough in lane 2). Excess oligo was successfully removed as shown in SYBR Gold DNA staining but the oligo-conjugated IgG failed to elute at low pH (lane 6). Most IgG remains on beads (lane 9).
Rabbit IgG (anti beta-tubulin; 80713-1-RR) was conjugated with NHS-dyes (lane 1 respectively). Post-conjugation clean-up was done usingCleanAb Kit (agarose) for Rabbit IgG (lanes 2-4). At least 95% of excess dye was successfully removed as evident from the faint excess dye band(lane 4). The conjugated IgG was eluted with high recovery (>70%) at neutral pH (lane 4). Desalting was done with Zeba spin desalting columns to exchange buffer after elution (lane 6), successfully removing all residual dye (buffer exchange is only recommended for long term storage or if required for downstream assays). No IgG remains on beads (lane 7). Note: the CleanAb Elution Buffer can interfere with SDS gels, which can make the CleanAb elution appear smeary.
Mouse IgG1 (anti Lamin B1; 66095-1-Ig) was conjugated with NHS-dyes (lane 1 respectively). Post-conjugation clean-up was done usingCleanAb Kit (magnetic agarose) for Rabbit IgG (lanes 2-4). At least 95% of excess dye was successfully removed as evident from the faint excess dye band (lane 4). The conjugated IgG was eluted with high recovery (>70%) at neutral pH (lane 4). Desalting was done with Zeba spin desalting columns to exchange buffer after elution (lane 6), successfully removing all residual dye (buffer exchange is only recommended for long term storage or if required for downstream assays). No IgG remains on beads (lane 7). Note: the CleanAb Elution Buffer can interfere with SDS gels,which can make the CleanAb elution appear smeary.
CleanAb vs Protein A
Both CleanAb and Protein A/G can efficiently remove excess DNA oligo or dye. However, CleanAb Kits demonstrate superior performance compared to Protein A across three critical parameters: binding affinity, elution conditions, and recovery yield.
Superior binding performance
CleanAb Beads demonstrate high and consistent affinity and capacity compared to Protein A, as evidenced by the absence of conjugated IgG in the flowthrough fraction. The enhanced binding capacity and consistent affinity towards all IgG subtypes not only increase yield but also improve reproducibility, ensuring consistent purification.
Gentle, native elution
Unlike Protein A, which requires acidic elution conditions that can compromise antibody integrity, CleanAb Beads allow efficient elution at native pH 7.5. This mild, physiologically relevant environment preserves protein conformation and biological activity - making CleanAb ideal for sensitive antibody-conjugates and downstream application. Reduced exposure to low pH also minimizes aggregation, denaturation, and loss of target molecules.
Maximized recovery
CleanAb Beads deliver high recovery rates of 60 - 90%, translating to significantly less product loss compared to Protein A. The optimized bead chemistry and elution profile ensure efficient release of bound conjugates with minimal residual binding. In contrast, Protein A frequently exhibits incomplete elution, leaving valuable material behind on the beads. The result: cleaner samples, higher yields, and more reliable downstream performance.
Note: Find more validation data on the respective product pages.
Desalting
CleanAb Kits efficiently remove excess ssDNA oligos or dyes and enable elution under gentle, native pH 7.5 conditions, preserving antibody functionality and conjugate integrity.
When to perform a buffer exchange
The CleanAb Elution Buffer contains reagents that can interfere with PAGE analysis or affect concentration measurements. If precise quantification is required for downstream application, a buffer exchange is recommended. Buffer exchange is also recommended for long-term storage, as long-term stability of conjugated antibodies in the elution buffer may vary depending on the antibody. Alternatively, the eluate can be stored at -80°C for long term.
Note: While buffer exchange may lead to minor sample loss, the resulting product remains fully functional, as demonstrated by the IF comparison shown on the right. Adjust final dilutions as needed for optimal performance. Buffer exchange columns are not included in the kit.
Immunostaining of HeLa cells with mouse anti-B23/NPM1 antibody (60096-1-Ig, 1:100, magenta), chemically conjugated to a 15-nt DNA oligonucleotide and purified using CleanAb beads. Detection was performed by hybridization with an imaging DNA strand labeled with ATTO643. Nuclei were counterstained with DAPI (yellow). Direct comparison of CleanAb product and desalted product demonstrates functionality of both products.
Custom solutions for high throughput
CleanAb-Tips
Contact us to discuss custom solutions for liquid handling systems and pre-filled pipette tips.
Large Scale CleanAb
Contact us for bulk orders or anything related to high throughput clean-ups.
Tech Support for CleanAb
Contact us for technical support.
Frequently Asked Questions
Where can I find detailed protocols for oligonucleotide or dye removal workflows with CleanAb?
You can find the protocols for the agarose kits and magnetic agarose kits on the product pages.
Do CleanAb Beads for Rabbit IgG and CleanAb Beads for Mouse IgG bind all rabbit or mouse antibodies, respectively?
CleanAb Beads for Rabbit IgG and CleanAb Beads for Mouse IgG both use kappa light chain-specific ligands and do not bind antibodies with lambda light chains. For both mouse and rabbit IgG, the vast majority of antibodies (>95%) have kappa light chains, while lambda light chain antibodies represent only a small minority. Therefore, CleanAb Beads are compatible with most rabbit or mouse IgG antibodies, respectively. If you are unsure which light chain your antibody carries, we recommend checking the manufacturer's documentation or contacting our technical support team before use.
Why do I have conjugated IgG in my flowthrough?
If you find that binding of your conjugated IgG after NHS conjugation is not working as expected, adjusting the molar ratio of your target molecule versus IgG might be necessary. Over-labeling, meaning too much oligo or dye, can negatively affect binding efficiency by blocking the binding site of the Fab-binding protein.
Important note: It is always advisable to determine the concentration of the IgG before performing a conjugation reaction. Concentrations stated by the supplier might not always be accurate. This helps avoid over-labeling of the IgG.
How much beads do I need to use?
The protocol suggests defined bead volumes per antibody amount. These volumes can be scaled up linearly. If you need to clean up less antibody, volumes can be reduced, although this might result in lower recovery rates.
Which conjugation chemistries are compatible with CleanAb Kits?
CleanAb Kits are compatible with antibody conjugates generated using the most common conjugation chemistries, including NHS ester-amine coupling, maleimide-thiol coupling, and click chemistry reactions.
Do CleanAb Kits remove only free oligonucleotides and dyes, or can they also remove unconjugated antibodies?
Because the beads bind the antibody itself, they capture both conjugated and unconjugated antibodies, while free oligonucleotides, dyes, and other non-bound components are removed during the washing steps.
Can CleanAb Kits be used to purify nanobodies or antibody fragments?
CleanAb Beads bind to the Fab region of antibodies in a species-specific manner. Based on this binding mechanism, CleanAb Kits are compatible with Fab and F(ab')2 antibody fragments, and can be used for Fab and F(ab')2 purification after enzymatic digestion using papain or pepsin.
They are not suitable for purification of alpaca-derived antibody fragments such as VHHs.
Can CleanAb Kits be used for removal of excess dye or unwanted buffer components?
Yes, CleanAb Kits can be used after conjugation of antibodies with small molecule dyes or to remove other unwanted buffer components like BSA, TRIS, sodium azide etc.
How pure is the product after elution from the beads?
CleanAb Kits remove more than 95% of excess dye and near 100% of excess oligo, as validated by HPLC. Doing the additional buffer exchange step usually removes any residual dye that remains in the eluate after elution from the beads.
Are CleanAb Kits suitable for large-scale or high-throughput antibody-conjugate purification workflows?
Yes, CleanAb Kits are suitable for large-scale and high-throughput antibody-conjugate purification workflows. Please contact us to discuss tailored solutions.
What are the advantages of CleanAb Kits compared to size exclusion chromatography or spin filtration?
Spin filtration can lead to antibody loss and may require multiple wash cycles to efficiently remove unreacted oligonucleotides. Size-exclusion chromatography can dilute samples and may require method optimization to achieve optimal recovery and separation.
In addition, not every laboratory has access to chromatography equipment. CleanAb Kits are designed to deliver a straightforward workflow with high, reproducible antibody recovery and efficient removal of excess oligonucleotides or dyes.
Should I do post-conjugation purification or on-bead conjugation with CleanAb Kits?
CleanAb Kits are compatible with both post-conjugation purification of antibody conjugates and on-bead conjugation workflows, and both approaches have been shown to deliver reliable, reproducible results. The main advantage of the on-bead conjugation workflow is that the antibody is first captured on the beads, allowing incompatible buffer components such as Tris, BSA, glycine, or other amine-containing additives to be removed before conjugation. This helps minimize interference with the conjugation chemistry and saves an extra step before conjugation.
For NHS ester-based conjugation specifically, however, we recommend the post-conjugation purification workflow. This is because NHS esters can react with functional groups on the immobilized ligand as well, reducing conjugation efficiency by consuming part of the labeling reagent. If the on-bead workflow is used with NHS chemistry, increasing the amount of oligo or dye may help compensate for this effect.