Conjugation kits

Conjugation kits simplify conjugation and are therefore an excellent option for those without chemical expertise.

The development of conjugation kits has simplified the traditionally complex conjugation processes, offering an excellent option for those without access to the expertise required to execute and optimize the complex methods described above. Most importantly, this option can prove helpful if you can’t find the appropriate combination of antibody and label readily available as an “off the shelf” product.

Conjugation kits, such as Lightning-Link®, use a simple, three-step process whereby the antibody to be labeled is added into a vial of a lyophilized mixture containing the desired label. The conjugated antibody is then ready to use without the need for post-conjugation purification steps, removing the risk of antibody loss. Figure 5 illustrates the Lightning-Link® conjugation kit process.

The advantages of this method include reproducibility and scalability, with conjugation kits that work with 10 µg–100 mg of antibody. Also, the kits’ ability to form antibody-label conjugates at a neutral pH in just three steps reduces the amount of hands-on bench time. Furthermore, the range of more than 45 different labels, as in Lightning-Link® kits, allows flexibility in the choice of application and multiplex panel development.

Download our conjugation application guide

https://go.myabcam.com/conjugation-guide

520

The Lightning-Link® kit labeling method.

Figure 5. The Lightning-Link® kit labeling method.

Four key considerations to generate high-quality conjugates

The conjugation efficiency of most conjugation chemistries, especially NHS-ester-based methods, relies on several factors, including your application, antibody purity and concentration, and buffer constituents.

To generate a high-quality conjugated antibody, follow these four steps:

Select the best label for your application: The choice of the label will largely depend on the final application. Table 2 lists labels and their most common applications.
Make sure your antibody is purified: Only purified antibodies are suitable for conjugation (ie, not crude serum or tissue culture supernatant).
Check the concentration of your antibody: the antibody should be at least 1 mg/mL .
Confirm the antibody is in a suitable buffer: certain buffer constituents can be incompatible with the labeling reaction (see table 4)

To achieve the best results with antibody conjugation, we recommend choosing an antibody in a buffer formulation free from glycerol, BSA, and sodium azide. These additives and other components that can interfere with the conjugation reaction will hinder the antibody’s labeling efficiency and result in poor-quality conjugated antibodies.

Abcam offers carrier-free antibodies specifically designed for antibody labeling. Carrier-free antibodies are conjugation-ready because they are provided in the most suitable buffer formulation and concentration. Choosing a carrier-free antibody will save you time and money due to unnecessary antibody loss post-purification, concentration, or buffer exchange.

If your antibody does not meet the requirements specified above, you can easily purify or concentrate your antibody using our range of purification and concentration kits.

Table 4. Buffer formulation components that can interfere with the conjugation reaction and purification kits

Unconjugated antibody properties

Compatibility

Notes

Purified antibody

Yes

Antibody in ascites fluid, serum, hybridoma or tissue culture media

Serum Antibody Purification Kit (Protein A) (ab109209) and Serum Antibody Purification Kit (Protein G) (ab128751) are suitable for purifying IgG fractions from serum and ascites fluid

Antibody concentration

> 1 mg/ml

The Antibody Concentration and Clean-Up Kit (ab102778) can be used to concentrate your antibody

Use the BCA Protein Assay Kit II (ab287853) to check your antibody concentration after you have removed any buffer components that could interfere with the concentration measurement

6.5 – 8.5

Amine free buffer (e.g. MES, MOPS, HEPES, PBS), NaCl, Borate buffer, Chelating agents (e.g. EDTA), Sugars,

Yes

BSA

Only at < 0.1%, w/v

Avoid if intending to use conjugated antibodies for IHC; BSA may cause background staining. The BSA Removal Kit (ab173231) could be used.

Sodium azide

Only at < 0.1%, w/v

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.

Glycerol

≤ 50 %, v/v

Use only at concentrations up to 50%

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

The Antibody Concentration and Clean-Up Kit (ab102778) can be used to remove any excess Tris from the buffer.

Glycine

The Antibody Concentration and Clean-Up Kit (ab102778) can be used to remove Glycine from the buffer.

Thiomersal / Thimerosal, Merthiolate, Proclin 300, Nucleophilic components (Primary amines e.g. amino acids or ethanolamine and thiols e.g. mercaptoethanol or DTT)

Please note that the individual 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.