Lightning-Link® Antibody Conjugation Kits - frequently asked questions (FAQs)
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Outline
Our antibody conjugation kits allow you to add fluorophores, chromogens, enzymes, biotin or other labels to your antibody. Although designed primarily for IgG conjugation, our kits allow you to label any biomolecule of your choice with some adjustment.
General information |
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General information about Antibody conjugation and Lightning-Link® Conjugation Kits
Which label should I use?
We provide quick and easy-to-use conjugation kits for over 45 labels including fluorescent and tandem dyes, proteins and enzymes that can be found here.
The choice of label primarily depends upon the application in which the antibody will be used.
Immunoassay | Commonly used labels |
ELISA | |
Flow cytometry | Alexa Fluor® Dyes, DyLight® Dyes, FITC, PE, PerCP, APC, and tandem dyes |
IHC |
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ICC | |
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How does Lightning-Link® conjugation work?
The labeling chemistry targets primary amines present in lysines and at the N-terminus of a protein. All antibodies have multiple free amine groups and most proteins have lysine and/or alpha-amino groups. The antibody or biomolecule simply needs to be pipetted into a vial of lyophilized mixture containing the label of interest, and incubated for around three hours (Lightning-Link® range) or 15 minutes (Lightning-Link® Fast range).
Despite the apparent simplicity of its protocol, the Lightning-Link® Conjugation process is sophisticated and quickly generates reproducible conjugates with no loss of antibody, saving you valuable time and resources.
What can be labeled?
As long as lysine residues or free reactive primary amines are present, primary and secondary antibodies as well as proteins, peptides and other biomolecules can be labeled with the Lightning-Link® Conjugation Kits.
The kits have been optimized for labeling IgGs, so for labeling non-antibody molecules, we would recommend optimizing the protocol by adjusting the amount of material added to the conjugation vial. This will help account for molecular weight differences between different types of molecules, as well as lysine residue content. This should be done without changing the total volume added to the vial, so that the conjugation efficiency is not decreased.
For more information on conjugating non-antibody molecules, read our advanced guide.
What recovery can be expected?
With Lightning-Link® Conjugation Kits, the entire antibody labeling reaction is contained within one tube and there are no separation steps involved. This means that 100% of antibody is retained at the end of the conjugation process. The conjugation process does not trigger antibody aggregation, and it is carried out at a physiological pH. Once the reaction is complete, you can usually use the conjugated antibody straight away without further purification steps.
What's the difference between Lightning-Link® and Lightning-Link® (Fast)?
Both types of kits can generate conjugates in 20–30 seconds hands-on time throughout the entire procedure.
However, the conjugation and quenching incubation times are shorter for Lightning-Link® (Fast) kits: only 15 minutes in total against three hours for the Lightning-Link® range.
The final quality of the conjugate will be the same.
Can I conjugate the same antibody with two different Lightning-Link® labels?
No. Lightning-Link® Conjugation Kits are designed for labeling antibodies with one type of label (single labeling). All kits target the same chemical groups on the antibody (free amines). Therefore, in a double-labeling experiment, the two different labels would compete for the same sites. We cannot guarantee the success of this kind of experiment.
Can I get custom-sized kits?
A range of kit sizes are available for purchase from the datasheet. If you want a size that’s not listed, get in touch with us to discuss your requirements. We can offer custom-sized kits up to 100 mg.
Buffers, materials, and conditions of the conjugation reaction:
The conjugation reaction using Lightning-Link® Conjugation Kits involves the formation of a covalent bond between lysine groups and the label. There are certain buffers and additives that can interfere or compete with this reaction, so it is important to be aware of these before proceeding to conjugation.
Do I need to purify the antibody prior to conjugation?
Yes. The antibody labeling chemistry involves free amine groups. Most proteins/peptides have lysine and/or alpha-amino groups, therefore, any protein/peptide present in the solution will also be labeled. We recommend purifying your antibodies before performing the conjugation or using purified antibodies. Ascites fluid, serum or hybridoma culture media can interfere with conjugation and should be avoided.
Our range of Antibody Purification kits allows simple and quick antibody purification and is fully compatible with Lightning-Link® Conjugation Kits. Find out more information in our antibody purification and concentration kits guide.
Which buffers are suitable?
The table below lists which buffers additives are suitable for use with our conjugation kits, and what can cause interference.
If you want an antibody formulation that’s free from any interfering additives, check out our carrier-free RabMab® antibodies, which can be used straight away with our conjugation kits.
Buffers, additives and conditions | Compatibility | Notes |
Purified antibody | ✓ Yes | |
| Antibody in ascites fluid, serum, hybridoma or tissue culture media | X No | |
| pH | 6.5 – 8.5 | |
| Amine free buffer (e.g. MES, MOPS, HEPES, PBS) | ✓ Yes | |
| Non-buffering salts (e.g. NaCl) | ✓ Yes | |
| BSA | Only at < 0.1% | Use only at concentrations <0.1%. Avoid if intending to use conjugated antibodies for IHC; BSA may cause background staining. |
| Sodium azide | Only at < 0.1% | Use only at concentrations <0.1%. Remove any sodium azide if labeling with HRP, as it is an irreversible inhibitor of HRP activity. The Antibody Concentration and Clean-Up Kit (ab102778) can be used for this. |
| Chelating agents (e.g. EDTA) | ✓ Yes | |
Glycerol | ≤ 50 % | Use only at concentrations up to 50 % |
| Sugars | ✓ Yes | |
| Gelatin | Only at < 0.1% | Use only at concentrations <0.1% Avoid if intending to use conjugated antibodies for IHC; Gelatin may cause background staining. |
| Tris | < 50 mM | Use only at concentrations <50mM The Antibody Concentration and Clean-Up Kit (ab102778) can be used to remove any excess Tris from the buffer. |
| Glycine | X No | The Antibody Concentration and Clean-Up Kit (ab102778) can be used to remove Glycine from the buffer. |
| Thiomersal / Thimerosal | X No | |
| Merthiolate | X No | |
| Proclin 300 | X No | |
| Borate buffer | ✓ Yes | |
| Nucleophilic components (Primary amines e.g. amino acids or ethanolamine and thiols e.g. mercaptoethanol or DTT) | X No |
Please note that, individually, the concentrations shown should not affect the conjugation reaction. However, in combination with additional compounds that are not recommended above a certain concentration, the reaction may be affected.
How do I remove additives from the antibody storage buffer?
Our antibody concentration and purification kits remove additives with ease and provide a ready-to-use antibody solution fully compatible with Lightning-Link® Conjugation kits.
Alternatively, if you want an antibody formulation that’s free from any interfering additives, check out our carrier-free RabMab® antibodies, which can be used straight away with our conjugation kits.
What is the optimal starting concentration for the antibody?
Please refer to the relevant datasheet and protocol for recommended antibody concentrations.
For the majority of kits, optimal conjugates are normally generated using antibodies at 1 mg/mL. However, we specify a range of acceptable starting concentrations in the protocol booklet; concentrations within this range will still generate quality conjugates.
If your starting antibody concentration falls outside the recommended range, we’d suggest concentrating or diluting the antibody as appropriate.
At what temperature can I carry out the conjugation reaction?
The conjugation reaction must be performed at room temperature.
Please note, the reaction cannot be carried out at 4°C. If the incubation temperature is lowered, the conjugation efficiency will be reduced.
What is the best way to check conjugation success?
Our range of conjugation check kits allows you to confirm the conjugation of an antibody in one easy step, without the need for any specialized or costly equipment. Please note that these kits are only suitable for the qualitative verification of IgG antibodies.
Alternatively, you can test conjugation success using a preliminary experiment in the application of interest.
Storage and use of the conjugated antibody:
Once the conjugation is complete using Lightning-Link® Conjugation Kits, the antibody can be used straight away without further purification steps for most applications.
How do I filter out the free label from the conjugated antibody?
Lightning-Link® conjugation kits are designed to give a low level of free label at the end of the reaction. Thus, no filtration steps are required. Any remaining free label would have its reactive groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
Do I need to desalt the final conjugated antibody?
No. In western blot, ELISA, IHC and other applications where one would normally use secondary antibodies, you can use the conjugated antibody straight away.
How do I store the conjugated antibody?
Depending on the antibody, it can be stored long-term at either -20ºC with glycerol, or at 4°C with suitable additives.
- All conjugates can be stored in 50% glycerol at -20°C which allows them to remain stable for 2 years. Please bear in mind that APC and R-PE conjugates should never be stored at -20°C on their own without glycerol. The dilution factor also has an effect, so storing the conjugate undiluted is recommended if possible.
- For long-term storage at 4°C, we recommend adding antimicrobial agents and/or stabilizers (e.g. azide, BSA, glycerol). A new conjugate can be stored for 12-18 months at 4°C, as long as the antibody will tolerate storage at this temperature. The exception to this is tandem conjugates, which are only stable for up to 3 months.
As the bond between the antibody and dye is covalent and very stable, the antibody is usually the least stable component of the conjugate.
At what concentration can the conjugated antibody be used?
For our conjugation kits, only the conjugation process itself is covered by our Abpromise™ guarantee. It’s important to carefully consider the conditions for using your conjugated antibody in your chosen application.
The optimal working concentration for the conjugate should be determined experimentally in the application of interest.
For help with optimizing antibody dilutions, see this page.
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.