ReadiView™ Biotin: All-In-One Biotinylation & Quantification of Biotin Labeling
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ReadiView™ Biotin: All-In-One Biotinylation & Quantification of Biotin Labeling

Fundamentals of Flow Cytometry

ReadiUse™ Lyophilized Phycobiliproteins

Buccutite™ Conjugation Technology

AssayWise Letters Vol. 8(1)

The biotin-streptavidin complex is one of the most popular tagging systems for the conjugation of biomolecules, such as proteins, lipids and nucleic acids, as well as that of synthetic molecules, such as fluorescent labels. It has found strong success in the area of sample preparation as a core part of many affinity purification systems, and plays a critical role in many detection systems for instruments such as microscopy and flow cytometry.

The widespread adoption of the biotin-streptavidin conjugation system is primarily due to the extraordinarily high binding affinity of streptavidin for biotin. This complex is one of the strongest known non-covalent interactions between a protein and ligand, and permits binding of biotinylated molecules to streptavidin conjugates in heterogeneous mixture. Bond formation is rapid, and able to withstand extremes in pH, temperature, organic solvents, and denaturing agents.

A key benefit to the biotin-streptavidin complex is its ability to significantly improve the detection sensitivity of any assay. This in large part is due to the tetrameric conformation of streptavidin, which enables streptavidin to bind four biotins per molecule with high affinity and selectivity. This multiplicity allows for amplification of weak signals, allowing for improved experimental results when testing for medium- to low- abundance targets in mammalian cells or tissues (Figure 1).



Figure 1. Shematic of signal amplification by biotin-streptavidin formation.


Biotinylation

Biotinylated proteins are routinely used in conjunction with streptavidin conjugates. Together, they form a sophisticated detection platform that is readily adaptable to a broad range of applications. A large factor that contributes to the versatility of the biotin-streptavidin system is the ability to conjugate streptavidin to a variety of reporter tags. For example, enzyme conjugates of streptavidin, such as HRP-streptavidin (Cat# 16920) or AP-streptavidin (Cat# 16921), are commonly used in immunoblotting, ELISA and in situ hybridization imaging applications. While fluorescently labeled streptavidin, such as iFluor™-streptavidin, are widely used in cell surface labeling, fluorescence activated cell sorting (FACs) and other fluorescence imaging applications.

Biotin is a 244 dalton vitamin that is present in trace amounts in all living cells. Besides having a strong affinity for streptavidin, biotin exhibits two characteristics that make it ideal for bioconjugate development. First, is biotin’s relatively small size. This minimizes any significant hindrance with a protein’s biological reactivity, and permits the biotinylation – labeling – of multiple biotin tags to a single macromolecule for maximum detection by streptavidin and its conjugates.

The second characteristic is the valeric acid side chain of the biotin molecule. This chain can be derivatized to incorporate various reactive groups used to biotinylate proteins without altering its binding affinity for streptavidin. This feature is critical as it allows for the development of various biotinylated reagents. These include, amine-reactive biotin succinimidyl ester and thiol-reactive biotin maleimide, as well as, ReadiView™ reactive biotins and ReadiLink™ Protein Biotinylation Kit (Cat #5521) which enable researchers to chemically label proteins, nucleic acids and other molecules in order make custom assay probes.


Biotinylation Reagents

Biotinylation can be performed enzymatically or chemically. Of the two methods, chemical biotinylation is more commonly utilized because it offers greater flexibility and can be performed in vitro and in vivo. Biotinylation reagents are typically comprised of three components, which include the biotin tag, a spacer arm and a reactive group. As previously mentioned, the valeric acid side chain of biotin is modified with reactive groups, this dictates target reactivity. Common protein targets for chemical modification include primary amines (-NH2), thiols (-SH), carboxyls (-COOH) and carbonyls (-CHO). Of the four, primary amines are the most targeted functional group because of their abundance and accessibility. Succinimidyl esters (SE) form stable amide bonds with primary amines and can readily be incorporated on biotin.

The distance between the reactive group and biotin tag can be adjusted using spacer arms of various lengths, which can increase the availability of biotin for streptavidin binding, reduce steric hindrance and increase reagent solubility. Spacer arm length is commonly regulated by hydrocarbons, PEG or disulfide bonds that allow for biotin cleavage. When determining the appropriate biotinylation reagent to use for a specific application and protein of interest carefully consider: (1) solubility, (2) spacer arm length, (3) functional group, and (4) degree of biotinylation.

Step 4, quantification of the degree of biotinylation, is useful in order achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Quantification of the degree of the biotinylation reaction can assist in optimizing a particular biotin-streptavidin system and ensure reproducibility in the biotinylation processes. However, it is challenging to quantitate biotin directly because its intrinsic absorption is difficult to distinguish from that of proteins and nucleic acids. Therefore, in order to quantitate biotinylation a separate method is required; this increases cost and hands-on-time.

The 4’-hydroxyazobenzene-2-carboxylic acid or HABA assay (Appendix A) is one such technique. Besides increasing cost and time, the HABA assay and similar streptavidin binding assays for measuring biotinylation suffer from numerous shortcomings, including poor sensitivity, requiring and consuming large sample volumes (up to 75 μg of labeled protein) and requiring external streptavidin-calibration curves.


Quantification of Biotinylation using ReadiView Biotin

To address these concerns, AAT Bioquest has designed ReadiView™ biotin (Figure 2). This family of reactive biotins includes five novel biotinylation reagents in various reactive formats: succinimidyl ester, maleimide, acid, hydrazide, and amine. The ReadiView™ biotin series has been optimized to streamline the biotinylation and quantification processes. Each ReadiView™ biotin reagent contains a specially designed Color Tag (CT) optimally positioned between two spacer arms. These spacer arms reduce steric hindrance and improve solubility, while the CT tag makes the degree of biotinylation readily quantifiable by calculating the absorption ratio of 280 nm/385nm. Additionally, the CT tag has minimal effect on the biotin binding affinity and minimal quenching effect on fluorophores used to label streptavidin.


Figure 2. ReadiView™ biotin conjugated with specially designed color tag (CT) for easy determination of biotinylation degree.


Table 1. ReadiView™ Biotin target reactivity.

Cat#Product NameFunctional Group Target
3059ReadiView™ biotin succinimidyl esterPrimary Amines (-NH2)
3058ReadiView™ biotin maleimideThiols or Sulfhydryls (-SH) 
3050ReadiView™ biotin acidAmines (-NH2)
3055ReadiView™ biotin hydrazideCarbonyls (-CHO)
3053ReadiView™ biotin amine Carbonyls (-CHO)


Protocol - ReadiView™ Biotin Succinimidyl Ester

The following is an overview of the protocol for ReadiView™ biotinylation and quantification of the biotinylation using ReadiView™ Biotin SE:

  1. Add 5 µl Reaction Buffer into target protein (50 µl)
  2. Add the protein solution into ReadiView™ Biotin SE
  3. Incubate at room temperature for 30-60 minutes
  4. Purify the conjugate by spin column
  5. Read absorbance at 280 and 389 nm and calculate the DOS using the equation


Detailed protocol for the biotinylation and quantification process is as follows:

Running the conjugation reaction:

  1. Add the protein working solution into the vial of ReadiView™ Biotin SE stock solution (2 µL/vial), and mix them well by repeatedly pipetting for a few times or vortex the vial for 2-5 minutes.
  2. Keep the conjugation reaction mixture at room temperature for 30 – 60 minutes.

    Note: The conjugation reaction mixture can be rotated or shaken for longer time if desired.

Preparation of the spin column for sample purification:

  1. Invert the provided spin column several times to re-suspend the settled gel and remove any bubbles.
  2. Snap off the tip and place the column in a washing tube (2 mL). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube.
  3. Centrifuge for 1 minute in a swinging bucket centrifuge at 1,000 x g to remove the packing buffer. Discard the buffer.
  4. Apply 1-2 mL 1X PBS (pH 7.2-7.4) to the column. After each application of PBS, let the buffer drain out by gravity, or centrifuge the column for 2 minutes to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3-4 times.
    Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.

Purification of the conjugates:

  1. Place the column in a clean Collecting Tube (1.5 mL). Carefully load the sample (20-100 µL, from Step conjugation reaction) directly to the center of the column.
  2. After loading the sample, add 1X PBS (pH 7.2-7.4) to make the total volume of 110 µL. Centrifuge the column for 5-6 minutes at 1,000 x g, and collect the solution that contains the desired biotin-labelled protein.

Quantification of the biotinylation process:

  1. Measure the absorption at 280 and 389 nm and calculate the number of biotin moieties displaced on your target protein.


Figure 3. Sensitivity assay performed using an ELISA on a known concentration of Mouse IgG antibody by applying Biotinylated Goat anti-mouse antibody, modified by ReadiView™ biotin succinimidyl ester (Cat# 3010), ReadiLink™ Protein Biotinylation Kit (Cat# 5521) or bought from vendor A, followed by Streptavidin-HRP (Cat# 16920). The colorimetric signal was generated using ReadiUse™ TMB Substrate Solution (Cat# 11003)


Product Ordering Information

Table 2. Product ordering information for iFluor™-streptavidin conjugates.

Cat#Product NameEx (nm)Em (nm)SizePrice
3059ReadiView™ biotin succinimidyl ester  5 mg$95
3058ReadiView™ biotin maleimide  5 mg$195
3050ReadiView™ biotin acid  25 mg$95
3055ReadiView™ biotin hydrazide  5 mg$195
3053ReadiView™ biotin amine  5 mg$195


Table 3. Product ordering information for iFluor™-streptavidin conjugates.

Cat#Product NameEx (nm)Em (nm)SizePrice
16950iFluor™ 350-streptavidin conjugate345442200 ug$75
16980iFluor™ 350-streptavidin conjugate3454421 mg$145
16952iFluor™ 405-streptavidin conjugate401420200 ug$75
16982iFluor™ 405-streptavidin conjugate4014201 mg$145
16955iFluor™ 488-streptavidin conjugate491514200 ug$75
16985iFluor™ 488-streptavidin conjugate4915141 mg$145
16956iFluor™ 514-streptavidin conjugate511527200 ug$75
16986iFluor™ 514-streptavidin conjugate5115271 mg$145
16957iFluor™ 532-streptavidin conjugate536560200 ug$75
16987iFluor™ 532-streptavidin conjugate5365601 mg$145
16959iFluor™ 555-streptavidin conjugate552567200 ug$75
16989iFluor™ 555-streptavidin conjugate5525671 mg$145
16962iFluor™ 594-streptavidin conjugate592614200 ug$75
16992iFluor™ 594-streptavidin conjugate5926141 mg$145
16965iFluor™ 633-streptavidin conjugate638655200 ug$75
16995iFluor™ 633-streptavidin conjugate6386551 mg$145
16966iFluor™ 647-streptavidin conjugate654674200 ug$75
16996iFluor™ 647-streptavidin conjugate6546741 mg$145
16968iFluor™ 680-streptavidin conjugate682701200 ug$75
16997iFluor™ 680-streptavidin conjugate6827011 mg$145
16970iFluor™ 700-streptavidin conjugate693713200 ug$75
16998iFluor™ 700-streptavidin conjugate6937131 mg$145
16973iFluor™ 750-streptavidin conjugate753779200 ug$75
16999iFluor™ 750-streptavidin conjugate7537791 mg$145
16958iFluor™ 546-streptavidin conjugate541557200 ug$75
16960iFluor™ 568-streptavidin conjugate568587200 ug$75


Appendix A: Common Methods For Measuring Biotinylation

HABA

The most common method to measure the degree of biotinylation of a sample is using 4'-hydroxyazobenzene-2-carboxylic acid (HABA) dye, which non-covalently binds to avidin in the absence of biotin. When bound to avidin, HABA exhibits an absorbance at 500 nm (A500), which is proportional to the amount of bound HABA. When a biotinylated sample is mixed with the HABA–avidin complex solution, the biotin displaces HABA for binding to avidin because the association constant of the avidin–biotin interaction is much greater than that for HABA–avidin (6 x 106 M-1). Because the absorbance of HABA is proportional to its binding to avidin, the amount of biotin present in the solution can be calculated based on the reduction in the A500 signal. The traditional colorimetric HABA assay requires lot of samples approx. (20 µL) and has sensitivity around 2 to 16 µM.


ELISA

Another method for the quantification of biotinylation is enzyme linked immunosorbent assay (ELISA). Biotinylated proteins are immobilized onto polystyrene microtiter plates, The free proteins are washed away and non-specific binding of proteins was blocked using the blocking buffer. The plate then will be incubated with streptavidin alkaline-phosphatase. The amount of streptavidin-enzyme conjugate retained in the wells is detected by yellow color produced by adding p-nitrophenyl phosphate which is measured at 405 nm wavelength. The amount of signal generated is directly proportional to the amount of biotinylated protein absorbed to the microtiter plate well surface.


SDS-PAGE

The presence of biotinylated and non-biotinylated proteins in a sample can also be estimated by binding of the sample to the streptavidin coated magnetic beads by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The test sample is incubated with the SA-coated magnetic beads for 30 minutes and the amount of non-bound can be compared to input. Now, this technique is based on the assumption that SA-coated beads are in excess to the amount of biotin attached to the protein. Also, there are chance of non-specific binding of the non-biotinylated protein to the beads.


References

  1. Hofmann K et al. (1982) Avidin binding of carboxyl-substituted biotin and analogues. Biochemistry 21:978–984
  2. Hirsch J et al. (2002) Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Analytical Biochemistry 308:343–357
  3. Sugawara K et al. (2005) Voltammetric homogeneous binding assay of biotin without a separation step using iminobiotin labeled with an electroactive compound. Anal Sci 21:897–900
  4. Hermanson GT (2008) Bioconjugate techniques, 2nd Edition. San Diego (CA): Academic Press
  5. Barat B, Wu AM (2007) Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum. Biomol Eng 24:283–91