Labeling Kits from AAT Bioquest bring Color to your Lab!

Written by Emily Locke

These topics await you:

1) Comparison of the Labeling Kits from AAT Bioquest

2) ReadiLink^TM Rapid Antibody Labeling Kits

3) BSA-compatible ReadiLink^TM xtra Rapid Antibody Labeling Kits

4) Buccutite^TM Protein to Protein Labeling Kits

5) Buccutite^TM HRP and Poly-HRP Antibody Labeling Kits

6) Buccutite^TM Rapid PE, APC, PerCP and Tandem Dye Antibody Labeling Kits

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Who doesn't like to sit outside with a glass of white wine spritzer and enjoy the sun? With a bit of luck, you might make a groundbreaking discovery during such a relaxed evening: In 1852, the Irish physicist and mathematician Sir George Gabriel Stokes observed the phenomenon of fluorescence for the first time over a glass of white wine. Sunlight filtered through a violet glass window struck a bottle of quinine water, whereupon blue light was emitted. This blue light passed through a glass of white wine, which filtered out the violet light from the window, leaving only the blue light from the quinine [1]. Stokes noticed this blue light and thus recognized the regularity that the light re-emitted by fluorescent substances has a longer wavelength than the light previously absorbed - a rule now known as the Stokes shift [2].

Nowadays, fluorescence has a variety of applications, especially in research: Fluorescent tags are used, for example, for live cell imaging, in flow cytometry or for immunofluorescence. For these applications, it is necessary to label antibodies or target proteins with a specific fluorescent tag. Through this so-called "labeling", the antibodies or proteins are permanently linked to the respective fluorescent dye. Besides fluorescent dyes, enzymes or haptens, such as biotin, can also be used as tags. These tags are used, among other things, in blotting, in ELISAs or for the isolation and purification of proteins [3].

A typical label consists of a binding group (linking group), possibly a connector (linker) and a detectable molecule (reporter) [4]. Our manufacturer AAT Bioquest, a specialist in fluorescence and luminescence technologies, offers an extensive range of kits for labeling antibodies and proteins as well as other biopolymers such as nucleic acids and carbohydrates. In the following, we would like to give you an overview of the leading product lines, including ReadiLink^TM and ReadiLink^TM xtra for easy labeling of antibodies with fluorescent dyes as well as Buccutite^TM for protein-protein conjugation.

ReadiLink^TM Rapid Antibody Labeling Kits

ReadiLink^TM labeling kits provide a fast and convenient method to label antibodies with fluorophores such as AAT's iFluor® or mFluor^TM dyes. They are suitable for multiple applications including fluorescence microscopy, multi-color imaging, multiplex flow cytometry, immunohistochemistry (IHC) and more. The ReadiLink^TM Rapid Antibody Labeling Kits offer the advantage that the produced product does not need to be purified and that the entire conjugation process takes only one hour. In addition, it is possible to label microgram quantities of antibodies, recovering 100% of the antibodies used [3].

The protocol for the labeling reaction involves only two simple steps: First, mix your prepared antibody solution at a concentration of 1 mg/mL with the ReadiLink^TM reaction buffer and add this mixture to your vial of pre-formulated markers provided in the kit (Fig. 1). These markers selectively bind to primary amines on the antibody to be labeled, such as lysine residues, allowing the preparation of covalently labeled conjugates. After an incubation step of 30 to 60 minutes, add the TQ^TM Quench Buffer (Fig. 1). This buffer eliminates both the need for a purification step and background interference. After an additional 10 minutes of incubation, the conjugated antibodies are ready to use [3].

Figure 1: Antibody labeling workflow with the ReadiLink™ Rapid Antibody Labeling Kit. With just a few simple steps, AAT's kit enables you to reliably label your antibodies: After preparing your antibody solution, add reaction buffer and mix the solution with the desired labeling dye (included in the kit). After 30 to 60 minutes of incubation, add the TQ^TM Quench Buffer and incubate the mixture for another 10 minutes. Et voilà: Your conjugates are ready to use [3]!

BSA-compatible ReadiLink^TM xtra Rapid Antibody Labeling Kits

AAT's ReadiLink^TM xtra Rapid Antibody Labeling Kit is an extension of the ReadiLink^TM Rapid Antibody Labeling Kits already described. It has exactly the same advantages (for example, the incredibly fast and simple protocol or the elimination of a purification step), but is also compatible with BSA and other stabilizing proteins or protein mixtures (e.g. gelatin) [3]. While such common buffer additives have to be removed beforehand with the ReadiLink^TM Rapid Antibody Labeling Kits, you can directly use your BSA-containing antibody solutions for the labeling reaction without any problems with this kit!

Buccutite^TM Protein to Protein Labeling Kits

While the ReadiLink^TM kits allow rapid labeling of antibodies with fluorescent dyes, you can use the Buccutite^TM labeling kits for protein-protein conjugation. This allows you to covalently label your proteins and antibodies with enzymes or phycobiliproteins [3]. In contrast to the conventional SMCC method, conjugation using Buccutite^TM technology is less demanding and extremely time-saving: whereas labeling via SMCC requires purification of the product and thus, the entire process takes four or more hours, conjugates using Buccutite^TM technology are ready to use within two hours without a purification step. In addition, the conjugates are extremely stable and therefore survive the thorough washing steps typically required for immunoassays (e.g. ELISA, IHC and Western blot) or flow cytometry [5].

• Buccutite^TM Rapid Protein Crosslinking Kit *Microscale Optimized for Crosslinking 100 ug Antibody per Reaction*

Buccutite^TM HRP and Poly-HRP Antibody Labeling Kits

Do you want to label your primary antibody with the detection enzyme horseradish peroxidase (HRP)? Then the Buccutite^TM HRP and Poly-HRP Antibody Labeling Kits are prefect for you! These kits allow labeling with HRP in under two hours and the conjugates obtained are ideal for various applications such as ELISA, IHC, Western blotting and tyramide signal amplification (TSA) imaging. In addition, the conjugates are extremely stable, making them suitable for long-term storage. They can be stored at +4 °C for up to 12 months [5].

HRP is a metalloenzyme that is used as one of the most common reporter enzymes in biochemistry. It is industrially isolated from horseradish - hence the unusual name - and enables the production of optically active hydroperoxides [6]. HRP conjugates alone are quite useless, as the peroxidase requires a substrate to produce a measurable signal. Depending on the substrate used, a chemiluminescence, fluorescence or a color reaction occurs. While the conversion of chemiluminescent substrates (e.g. luminol) generates by-products that can be detected using a luminometer, the conversion of a substrate for fluorescence (e.g. Amplite®) can be measured using a wide variety of fluorescence instruments. Dye-based substrates (e.g. ABTS, TMB) have been used for years, with the resulting color change quantified by an absorbance microplate reader. Due to their compatibility with a wide variety of substrates, HRP conjugates allow some flexibility in experimental design [5].

With AAT's Buccutite^TM Kits, you can label your antibodies with an HRP molecule within 2 hours. All you need to do is activate your antibody with the Buccutite^TM MTA linker included in the kit and incubate it for one hour with Buccutite^TM Folic Acid (FOL)-activated HRP - and your conjugates are ready to use (Fig. 2)!  Buccutite^TM HRP Antibody Labeling Kits are available in three different sizes optimized for labeling 25 µg, 100 µg, and 1 mg of antibody per labeling reaction. In addition, AAT offers Buccutite^TM Poly-HRP Antibody Labeling Kits that allow direct labeling of antibodies with HRP polymers. These conjugates achieve a higher level of sensitivity and a better signal-to-noise ratio. Thus, they are ideal for applications where the target protein is present in low quantity or the sample volume is limited [5].

Figure 2: Workflow of the Buccutite^TM Peroxidase (HRP) Antibody Labeling Kit. The antibody is first activated by a 30-minute incubation step with the MTA linker included in the kit. Thereafter, the antibody, now coupled to the linker, is incubated with the FOL-activated HRP enzyme for 60 minutes and is then ready to use [5].

Buccutite^TM Rapid PE, APC, PerCP and Tandem Dye Antibody Labeling Kits

However, Buccutite^TM technology not only allows you to conjugate HRP to your antibodies - you can also label them with phycobiliproteins, such as phycoerythrin (PE), allophycocyanin (APE) or the peridinin-chlorophyll protein (PerCP). These so-called chromoproteins consist of a protein part as well as a chromophoric group [7] and are capable of generating more intense fluorescent signals than any commercially available synthetic fluorophore [5]. They are particularly used for applications that require high sensitivity but low photostability, such as flow cytometry, fluorescence activated cell sorting (FACS), or immunophenotyping. Moreover, phycobiliproteins allow multiparametric analyses: by labeling them with small organic fluorophores, such as FITC (fluorescein isothiocyanate), PE, PE-Cy5, or PE-Cy7, tandem dyes with a very large Stokes shift (back to the white wine ?) are generated, which show excellent spectral separation despite the same excitation source [5]. In AAT's catalog "PE & APC Tandem Dyes" you can learn more about these innovative dyes!

The protocol for conjugation of phycobiliproteins to an antibody is as simple as for labeling with HRP (Fig. 3). After only two steps the conjugate is ready to use, purification is not necessary with this kit either. You can also choose between three different sizes depending on the amount of antibody per labeling reaction (25 µg, 100 µg or 1 mg) and store the prepared conjugates at +4 °C for up to 12 months [5].

Figure 3: Workflow of the Buccutite^TM Rapid Antibody Labeling Kit for phycobiliproteins. The antibody is first activated by a 30-minute incubation step with the MTA linker included in the kit. Thereafter, the antibody, now coupled to the linker, is incubated with the FOL-activated phycobiliprotein (e.g., PE, APC, PerCP, or a tandem dye) for 60 minutes and is then ready to use [5].

*Size for all kits: 2 Labelings

Feel free to take a look at AAT's labeling brochure for more details about the available dyes for protein labeling. You can also read about classic labeling fluorescent dyes in this blog article.

Fluorescent or luminescent technologies in general might be of interest to your research? Then browse through AAT Bioquest's other products and bring color to your lab!

Sources

[1] Stokes, G.G. XXX. On the change of refrangibility of light. Phil. Trans. R. Soc. 142: 463-562 (1852).

[2] https://www.edinst.com/blog/what-is-the-stokes-shift/, 12.08.2023.

[3] https://www.aatbio.com/catalog/antibody-and-protein-labeling, 12.08.2023.

[4] https://de.wikipedia.org/wiki/Molek%C3%BClmarkierung, 12.08.2023.

[5] https://www.aatbio.com/catalog/bioconjugation-protein-to-protein, 12.08.2023.

[6] Veitch NC. Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry. 65(3): 249-59 (2004).

[7] Aráoz R, Lebert M, Häder DP. Electrophoretic applications of phycobiliproteins. Electrophoresis. 19(2): 215-9 (1998).

Preview Image: https://www.aatbio.com/catalog/antibody-and-protein-labeling