Biotin and Streptavidin

Biotin-Streptavidin Conjugation System

The widespread adoption of the biotin-streptavidin conjugation system is primarily due to two factors. The first is the relatively small size of the biotin and streptavidin molecules themselves, which allows for extensive binding to biologically active macromolecules, such as antibodies, without impedance to their functions (i.e. an antibody’s binding site). The second is the specificity with which biotin and streptavidin bind to each other, as well as the strength of the subsequent bond, which allows for powerful, stream-lined bioassay applications. Streptavidin tetramers have an extraordinarily high binding affinity for biotin with a dissociation constant (K_d) of approximately ∼10^-14 mol/L. This tight and specific binding is rapid and able to withstand extremes in pH, temperature, organic solvents, and denaturing reagents.

Figure 1. Illustration of the Streptavidin-Biotin interaction. The multivalent properties of streptavidin allow it to bind up to four biotin molecules with a high degree of affinity. Biotin is typically conjugated to an enzyme, antibody or target protein.

Figure 2. Immunofluorescent stain of α-tubulin in HeLa cells. α-tubulin in fixed and permeabilized HeLa cells were labeled with rabbit anti-tubulin primary antibody, followed by incubation with biotinylated goat anti-rabbit IgG (H&L) (Cat No. 16794), and then visualized with iFluor™ 555-streptavidin conjugate (Cat No. 16989).

Table 2. Available Products for Biotinylated Secondary Antibodies

Biotinylated Nucleotides

Biotin-modified nucleoside triphosphate analogs, such as dUTP and dCTP, can be enzymatically incorparted into DNA or RNA fragments for use in fluorescence in situ hybridization (FISH), DNA arrays, microarrays and other hybridization techniques. Standard enzymatic non-radioactive DNA labeling reactions including 3”-end labeling, cDNA labeling, nick translation, PCR and random prime labeling. Each biotinylated nucleotide contains either an 11-, 14-, 16- or 20-atom spacer between the biotin and its attachment point on the nulceotide. This facilitates its detection and signal amplification by fluorophore and enzyme streptavidin conjugates or agarose and magnetic beads.

Biotinylated Amino Acids

Biotinylated peptides are suitable for immunodetection studies and biomedical screening assays that require immobilization onto streptavidin or avidin coated microtitre plates, beads or membranes. Biotin can be linked to the N-terminus of peptides or to the side chains of lysine (Lys), glutamic acid (Glu) or aspartic acid (Asp) during Fmoc solid phase synthesis. Incorportion of a PEG-spacer in both FMOC-Glu(biotinyl-PEG)-OH and FMOC-Asp(biotinyl-PEG)-OH reduces steric hindrance between target peptide and streptavidin, leading to better biotin binding. The hydrophilic nature of the PEG-spacer minimizes non-specific interactions and improves the solubility of the reagent and biotinylated peptide.

Reactive-Biotin Derivatives

Reactive biotin derivatives contain reactive moieties, such as succinimidyl esters or maleimides, that facilitate the labeling of biotin onto proteins, antibodies and other macromolecules. When selecting a reactive biotin derivative, consider the following:

• Funtional group targeting – specific reactive moieties target and bind to their respective functional groups for targeted or non-selective biotinylation of proteins and other macromolecules (e.g. succinimidyl esters target primary amines and maleimides target thiol or sulfhydryl groups).
• Solubility – this influences biotinylation of the target protein or macromolecule.
• Spacer Arm Length (PEG) – enhance detection senstivity of the target protein and reduces steric hindrance

ReadiView™ Biotinylation Reagents

Determining a protein’s degree of biotinylation is quite challenging because the intrinsic absorption of biotin is difficult to discern from proteins and nucliec acids. To facilitate this determination, we offer a series of chromophoric reactive boitin derivatives, ReadiView™ biotin. These novel biotinylation reagents contain a specially designed ‘Color Tag (CT)’ optimally positioned between two spacer arms. These spacer arms reduce steric hindrance and improve water solubility, while the CT tag makes the degree of biotinylation readily quantifiable by calculating the absorption ratio of 280 nm/385 nm. The CT tag does not affect biotins complexation with streptavidin and has minimal quenching affect on fluorescent streptavidin conjugates. ReadiView™ biotin is availble in amine-reactive, thiol-reactive and click-chemistry formats.

Figure 3. ReadiView™ biotin succinimidyl ester (Cat No. 3059) conjugated with specially designed color tag (CT) for easy determination of biotinylation.

Enzyme-Labeled Streptavidin Conjugates

Figure 4. IHC detection of EpCAM in FFPE lung adenocarcinoma tissue. Human adenocarcinoma tissue sections were incubated with rabbit anti-EpCAM primary antibody followed by biotyinylated goat-anti rabbit IgG (H&L). The samples were then incubated with HRP-streptavidin, and the signal was developed using ReadiUse™ Stayright™ Purple.

Figure 5. Immunofluorescent stain of &alpha-tubulin in HeLa cells. &alpha-tubulin in fixed and permeabilized HeLa cells were incubated with rabbit anti-tubulin primary antibody, followed by incubation with biotinylated goat anti-rabbit IgG (H&L) (Cat No. 16794), and then visualized with iFluor™ 647-streptavidin conjugate (Cat No. 16966. Nuclei were counterstained using DAPI (Cat No. 17507.)

Table 10. iFluor-Streptavidin Conjugates For Imaging and Flow Cytometry

Note

1. Ext. Coeff. = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm^-1M^-1.
2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. N/D = Not determined.

mFluor™ Streptavidin Conjugates for Flow Cytometry

Table 11. mFluor™ Streptavidin Conjugates for Flow Cytometry

Streptavidin APC, PE, PerCP and Tandem Dye Conjugates for Flow Cytometry

Streptavidin PE, APC and PerCP conjugates are commonly used in applications that require high sensitivity, but not phostostability, primarily flow cytometry, FACS, immunophenotyping, and microarrays. The unusually bright fluorescence of phycobiliproteins is due to their unusually high extinction coefficients and quantum yields.

Tandem dye conjugates compromise of a donor phycobiliprotein (e.g. APC or PE) labeled to acceptor iFluor™ dyes that emit fluorescence at a longer-wavelength. Using fluorescence resonance energy transer (FRET), light absorbed by the the donor phycobiliprotein results in fluorescence emission of the acceptor. In flow cytometry, tandem dyes are ideally suited for multicolor parametric analysis of cells due to their exploitation of a single excitation source and their significantly large Stokes shifts.

Streptavidin-Xtra™ Conjugates

The optimal degree of labeling of Streptavidin-Xtra™ iFluor™ conjugates produce the brightest fluorescent streptavidin conjugates for the detection of biotinylated antibodies in cell imaging, flow cytometry and western blot applications. Streptavidin-Xtra™ iFluor™ conjugates image low-abundance targets with greater sensitivity and detail compared to traditional streptavidin Alexa Fluor® conjugates.

Advantages

• Bright and photostable fluorescence without significant self-quenching
• High siganl-to-noise ratios: 3∼5 fold signal improvement versus Alexa Fluor®
• pH insensitive fluroescence over a wide molar range

Figure 7. Comparison of HeLa cells stained with Streptavidin-Xtra™ IF488 and Streptavidin-Alexa Fluor® 488.HeLa cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.02% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 1 µg/mL α-tubulin Mouse mAb for 1 hour at room temperature. Biotinylated goat anti-mouse IgG (H+L) (Cat No. 16729) was used for detection α-tubulin and visualized with Streptavidin-Xtra™ iFluor 488 (Cat No. 46001) and Streptavidin-Alexa Fluor® 488. Cells were counterstained with Hoechst 33342 (Cat No. 17535).

Table 13. Streptavidin-Xtra™ Conjugates

Product Ordering Information