Molecular Probes

Section 7.6 — Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices

The high affinity of avidin for biotin was first exploited in histochemical applications in the mid-1970s. This egg-white protein and its bacterial counterpart, streptavidin, have since become standard reagents for diverse detection schemes. In their simplest form, such methods entail applying a biotinylated probe to a sample and then detecting the bound probe with a labeled avidin or streptavidin. These techniques are commonly used to localize antigens in cells and tissues and to detect biomolecules in immunoassays and DNA hybridization procedures (Section 8.5). In some applications, immobilized avidins are used to capture and release biotinylated targets. In addition to our important dye and enzyme conjugates of avidins and streptavidins, this section contains several products that can be used for the affinity isolation of biotin- and DSB-X biotin–conjugated molecules and their complexes with targets in cell and tissues. Our unique DSB-X biotin technology, which is described below, provides the most facile means available for reversing the strong interaction of biotin derivatives with avidins. The product lists in Chapter 4 contain all of our biotin derivatives and biotin conjugates, including biotinylation reagents, biotin-based tracers and biotinylated site-selective probes, as well as our important DSB-X biotin reagents and conjugates.

Binding Characteristics of Biotin-Binding Proteins

Avidin, streptavidin and NeutrAvidin biotin-binding protein each bind four biotins per molecule with high affinity and selectivity. Dissociation of biotin from streptavidin (S888) is reported to be about 30 times faster than dissociation of biotin from avidin (A887, A2667). Their multiple binding sites permit a number of techniques in which unlabeled avidin, streptavidin or NeutrAvidin biotin-binding protein can be used to bridge two biotinylated reagents. This bridging method, which is commonly used to link a biotinylated probe to a biotinylated enzyme in enzyme-linked immunohistochemical applications, often eliminates the background problems that can occur when using direct avidin– or streptavidin–enzyme conjugates. However, a few endogenously biotinylated proteins that have carboxylase activity are found in the mitochondria (, ); therefore, sensitive detection of biotinylated targets in cells requires the use of biotin-blocking agents to reduce this background. Our Endogenous Biotin- Blocking Kit (E21390, see below) provides the reagents and a protocol for this application. Nonspecific binding of avidin conjugates of enzymes to nitrocellulose can be blocked more effectively by adding extra salts to buffers rather than by adding protein-based blocking reagents.

High-purity unlabeled avidin (A887), streptavidin (S888), NeutrAvidin biotin-binding protein (A2666) and CaptAvidin biotin-binding protein (C21385) are available in bulk from Molecular Probes at reasonable prices. We also offer avidin specially packaged in a smaller unit size for extra convenience (A2667). Our avidin, streptavidin and deglycosylated NeutrAvidin biotin-binding protein each bind greater than 12 µg of biotin per mg protein. See below for a description of reversible binding of biotinylated targets with our CaptAvidin biotin-binding protein and other affinity matrices.

Avidin

Avidin (A887, A2667; Table 7.23; Avidin and NeutrAvadin(R) Biotin-Binding Proteins and Conjugates) is a highly cationic 66,000-dalton glycoprotein with an isoelectric point of about 10.5. It is thought that avidin's positively charged residues and its oligosaccharide component (heterogeneous structures composed largely of mannose and N-acetylglucosamine) can interact nonspecifically with negatively charged cell surfaces and nucleic acids, sometimes causing background problems in some histochemical applications and flow cytometry. Methods have been developed to suppress this nonspecific avidin binding. In some cases, avidin's nonspecific binding can also be exploited. For example, avidin and its conjugates selectively bind to a component in rodent and human mast cell granules in fixed-cell preparations and can be used to identify mast cells in normal and diseased human tissue without requiring a biotinylated probe.

Streptavidin

Streptavidin (S888, Table 7.23, Streptavidin and Fluorescent Conjugates of Streptavidin), a nonglycosylated 52,800-dalton protein with a near-neutral isoelectric point, reportedly exhibits less nonspecific binding than avidin. However, streptavidin contains the tripeptide sequence Arg–Tyr–Asp (RYD) that apparently mimics the Arg–Gly–Asp (RGD) binding sequence of fibronectin, a component of the extracellular matrix that specifically promotes cellular adhesion. This universal recognition sequence binds integrins and related cell-surface molecules. Background problems sometimes associated with streptavidin may be attributable to this tripeptide. We have particularly observed binding of streptavidin and anti-biotin conjugates to mitochondria in some cells (, ) that can be blocked with the reagents in our Endogenous Biotin- Blocking Kit (E21390, see below).

NeutrAvidin Biotin-Binding Protein

Molecular Probes provides an alternative to the commonly used avidin and streptavidin. Our conjugates of NeutrAvidin biotin-binding protein (A2666, Table 7.23, Avidin and NeutrAvadin(R) Biotin-Binding Proteins and Conjugates) — a protein that has been processed to remove the carbohydrate and lower its isoelectric point — can sometimes reduce background staining. The methods used to deglycosylate the avidin are reported to retain both its specific binding and its complement of amine-conjugation sites. NeutrAvidin conjugates have been shown to provide improved detection of single-copy genes in metaphase chromosome spreads.

CaptAvidin Biotin-Binding Protein: Reversible Binding of Biotinylated Molecules

CaptAvidin biotin-binding protein is our newest avidin derivative (C21385, Table 7.23, CaptAvidin Biotin-Binding Protein). Selective nitration of tyrosine residues in the four biotin-binding sites of avidin considerably reduces the affinity of the protein for biotinylated molecules above pH 9. Consequently, biotinylated probes can be adsorbed at neutral pH and released at pH ~10 (Figure 7.96). We use free biotin to block any remaining high-affinity biotin-binding sites that have not been nitrated. CaptAvidin agarose (C21386, see below) is particularly useful for separation and purification of biotin conjugates from complex mixtures. The biotin-binding capacity of CaptAvidin derivatives is at least 10 µg of free biotin per mg protein.

Secondary Detection with Avidins

Avidin, streptavidin and NeutrAvidin conjugates are extensively used as secondary detection reagents in histochemical applications (, ), FISH (Section 8.5, Figure 8.92), flow cytometry, microarrays (Section 8.5, Figure 6.42), blot analysis (Section 9.4, Figure 9.53) and immunoassays. These reagents can also be employed to localize biocytin, biotin ethylenediamine or any of our fluorescent biocytins — all of which are biotin derivatives commonly used as neuroanatomical tracers (Section 14.3). DSB-X desthiobiocytin and DSB-X biotin ethylenediamine (D20652, D30752; Section 14.3) are similar polar tracers that reversibly bind to avidin derivatives.

The following are commonly used methods for employing avidin, streptavidin, NeutrAvidin biotin-binding protein and CaptAvidin biotin-binding protein as secondary detection reagents:

• Direct procedure. A biotinylated or desthiobiotinylated primary probe such as an antibody, single-stranded nucleic acid probe or lectin is bound to tissues, cells or other surfaces. Excess protein is removed by washing, and detection is mediated by reagents such as our fluorescent avidins, streptavidins or NeutrAvidin biotin-binding proteins or our enzyme-conjugated streptavidins plus a fluorogenic (), chromogenic or chemiluminescent substrate. Enzyme conjugates of streptavidin are key reagents in some of our Tyramide Signal Amplification (TSA) Kits (Section 6.2; Table 6.1; Figure 6.10, Figure 6.11, ) and in several of our kits for ultrasensitive detection of proteins on blots (Section 9.4, Table 9.9).
• Capture and release. Our unique DSB-X biotin technology (see below) permits the fully reversible labeling of DSB-X biotin derivatives by avidin and streptavidin conjugates (Figure 7.100). Consequently, targets in cells and tissues or on blots labeled with DSB-X biotin conjugates of antibodies (Section 7.2, Table 7.11) or other DSB-X biotin reagents can initially be stained with fluorescent avidin or streptavidin conjugates, then the fluorescent staining can be reversed with D-biotin (B1595, B20656; Figure 7.100, ) and the sample restained with an enzyme-conjugated avidin or streptavidin derivative in conjunction with a permanent stain such as diaminobenzidine (DAB, D22187; ) or the combination of NBT and BCIP (N6495, B6492, N6547; Section 10.3).
• Bridging methods. A biotinylated antibody or oligonucleotide is used to probe a tissue, cell or other surface. This preparation is then treated with unlabeled avidin, streptavidin or NeutrAvidin biotin-binding protein. Excess reagents are removed by washing, and detection is mediated by a biotinylated detection reagent such as a fluorescent biotin or biocytin dye (Section 4.3), biotinylated R-phycoerythrin (P811, Section 6.4), biotinylated FluoSpheres microspheres (Section 6.5) or biotinylated horseradish peroxidase (P917) plus a fluorogenic, chromogenic or chemiluminescent substrate.
• Indirect procedure. An unlabeled primary antibody is bound to a cell followed by a biotinylated species-specific secondary antibody. After washing, the complex is detected by one of the two procedures described above. Our Zenon Biotin-XX and DSB-X Biotin Antibody Labeling Kits (Section 7.3, Table 7.14) permit the rapid and quantitative biotinylation of antibodies for combination with avidin–biotin detection methods.

Endogenous Biotin-Blocking Kit

Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes sometimes produce substantial background signals when avidin–biotin detection systems are used to identify cellular targets (, , ). Because biotin-based technologies can be so sensitive — particularly when using enzyme-amplified detection methods such as TSA — we recommend preblocking endogenous biotin present in cells with the reagents in our Endogenous Biotin-Blocking Kit (E21390). This kit provides streptavidin and biotin solutions in convenient dropper bottles and an easy-to-follow protocol (Endogenous Biotin-Blocking Kit). Sufficient material is provided for approximately one hundred 18 mm × 18 mm glass coverslips.

Image-iT FX Kits: All-in-One Kits for Fluorescence Imaging of Fixed Cells

Image-iT FX Kits

The Image-iT FX Kits (Table 7.6) provide some of our best secondary detection reagents and the supporting materials needed for optimal imaging of fixed cells and tissue sections:

• Alexa Fluor conjugates of streptavidin, goat anti–mouse IgG antibody or goat anti–rabbit IgG antibody deliver superior photostability and brightness (Section 7.2, Table 7.7)
• ProLong Gold antifade reagent reduces photobleaching (Figure 23.25, , ; see Section 23.1 for more details)
• Image-iT FX signal enhancer improves the signal-to-noise ratio (, , )
• A sample pack of two CultureWell chambered coverglasses makes sample processing more convenient (Figure 23.35, see Section 23.3 for more details)

Each Image-iT FX Kit provides sufficient materials to perform 50–100 assays. Furthermore, the components of each kit are available separately (Alexa Fluor streptavidins, Table 7.23; Alexa Fluor secondary antibodies, Table 7.1, Section 7.2; ProLong Gold antifade reagent, P36930, Section 23.1; Image-iT FX signal enhancer, I36933, Section 7.2; CultureWell chambered coverglasses, C37000, C37005, Section 23.3) for flexibility in experimental design.

Image-iT FX Signal Enhancer

By efficiently blocking nonspecific interactions of a wide variety of fluorescent dyes with cell and tissue constituents, the Image-iT FX signal enhancer (I36933, Section 7.2) dramatically improves the signal-to-noise ratio of immunolabeled cells and tissues, allowing clear visualization of targets that would normally be indistinguishable due to background fluorescence (, , ). Background staining seen with fluorescent conjugates of streptavidin, goat anti–mouse IgG antibody or goat anti–rabbit IgG antibody is largely eliminated when Image-iT FX signal enhancer is applied to fixed and permeabilized cells prior to staining. Image-iT FX signal enhancer may also effectively prevent nonspecific staining that is typically blocked with 1–2% BSA or 10% serum treatment, in some cases eliminating the need for another step in the staining protocol.

Qdot Streptavidin Conjugates

Qdot streptavidin conjugates combine the unsurpassed photostability of Qdot nanocrystals with the highly specific binding properties of streptavidin. The large surface area afforded by the Qdot nanocrystal allows simultaneous conjugation of multiple streptavidin molecules to a single fluorophore. Advantages conferred by this approach include increased avidity for targets, the potential for cooperative binding in some cases and the use of efficient signal amplification methodologies. For example, combining biotin-functionalized products with the streptavidin labels allows for successive enhancements in signal via "sandwiching" (streptavidin/biotin/streptavidin, etc.) following an initial labeling step.

These powerful fluorescence detection reagents offer unique performance advantages in a wide variety of tissue labeling and flow cytometry experiments; they are efficiently excited using the 405 nm violet laser, and the Qdot nanocrystal fluorescence is extremely resistant to photobleaching. Not only can tissues stained with Qdot nanocrystals be observed for hours, but these stained tissues can be archived permanently; re-analysis of archived samples remains as quantitative as it was during the initial assay.

Our selection of Qdot streptavidin conjugates can all be excited by a single excitation source, enabling easy multicolor analysis of multiple targets or events in a single sample using color filtering to resolve the individual signals:

• Qdot 525 streptavidin conjugate (Q10141MP)
• Qdot 565 streptavidin conjugate (Q10131MP)
• Qdot 585 streptavidin conjugate (Q10111MP)
• Qdot 605 streptavidin conjugate (Q10101MP)
• Qdot 655 streptavidin conjugate (Q10121MP)
• Qdot 705 streptavidin conjugate (Q10161MP)
• Qdot 800 streptavidin conjugate (Q10171MP)
• Qdot Streptavidin Sampler Kit (Q10151MP)

Fluorescent Conjugates of Biotin-Binding Proteins

Fluorophore-Labeled Avidin, Streptavidin and NeutrAvidin Biotin-Binding Protein

Fluorescent avidin and streptavidin are extensively used in DNA hybridization techniques, immunohistochemistry (), MHC tetramer technology (Technical Focus: MHC Tetramer Technology) and multicolor flow cytometry. Molecular Probes' selection of avidin, streptavidin and NeutrAvidin conjugates keeps growing as we introduce new and improved fluorophores and signal amplification technologies (Table 7.23). We continue to provide avidin, streptavidin and NeutrAvidin conjugates of fluorescein (A821, S869, A2662), tetramethylrhodamine (S870, A6373), rhodamine B (S871) and Texas Red (A820, S872, A2665, S6370) dyes. However, we strongly recommend that researchers evaluate our many newer fluorescent conjugates:

• The green-fluorescent Alexa Fluor 488 () and Oregon Green () conjugates are not only brighter than fluorescein conjugates, but also much more photostable and less pH sensitive (Section 1.3; Figure 7.23, Figure 1.9, , ) (Product Highlight: The Alexa Fluor Dye Series — Peak Performance across the Visible Spectrum).
• Like the Alexa Fluor 488 conjugate, the green-fluorescent Alexa Fluor 500 () and Alexa Fluor 514 () streptavidin conjugates are far superior to fluorescein in both brightness and photostability, and they can be detected with standard fluorescein, Oregon Green dye or Alexa Fluor 488 dye filter sets. However, these Alexa Fluor conjugates are specifically designed to be detected simultaneously with other green fluorophores using instruments with the capacity to differentiate between fluorescence emission maxima <5 nm apart. The Alexa Fluor 500 dye can be optically separated from the Alexa Fluor 514 dye, and the Alexa Fluor 514 dye can be optically separated from both the Alexa Fluor 488 and Alexa Fluor 500 dyes. We also offer the yellow-green–fluorescent Alexa Fluor 532 streptavidin ().
• Other conjugates made with some of our brightest dyes include those labeled with our orange- and red-orange–fluorescent Alexa Fluor 546 (), Alexa Fluor 555 (), Alexa Fluor 568 () and Rhodamine Red-X () dyes, as well as those labeled with our red-fluorescent Alexa Fluor 594 (), Alexa Fluor 610 () and Texas Red-X () dyes. These conjugates are more fluorescent than traditional Lissamine rhodamine B and Texas Red conjugates (Figure 1.76, Figure 1.83), yet have similar excitation and emission maxima (Figure 1.74).
• Our Alexa Fluor 633 (), Alexa Fluor 635 (), Alexa Fluor 647 (), Alexa Fluor 660 (), Alexa Fluor 680 (), Alexa Fluor 700 () and Alexa Fluor 750 () conjugates of streptavidin have fluorescence that is not visible to the eye, but their absorption occurs at wavelengths that are easily excited by laser and laser diode light sources (Figure 1.24) and their fluorescence is easily detected by infrared light–sensitive detectors. Conjugates of the Alexa Fluor 555 and Alexa Fluor 647 dyes, in particular, have fluorescence that is superior to that of the spectrally similar Cy3 and Cy5 dyes (Figure 7.37, Figure 7.38), respectively, and their conjugates are more photostable than Cy3 and Cy5 conjugates (Figure 1.28). Furthermore, our Alexa Fluor 635 dye produces brighter protein conjugates than does the Alexa Fluor 633 dye because the absorption spectrum of the Alexa Fluor 635 dye does not split into two peaks upon protein conjugation, as do the absorption spectra of the Alexa Fluor 633, Cy5 and tetramethylrhodamine dyes (Figure 1.71).
• For blue-fluorescent labeling, we offer streptavidin and NeutrAvidin conjugates of the Alexa Fluor 350, Alexa Fluor 405, Marina Blue, Cascade Blue and Pacific Blue fluorophores. In side-by-side testing, our Alexa Fluor 350 streptavidin (S11249) displays significantly more fluorescence than AMCA streptavidin (Figure 7.31).
• The Alexa Fluor 430 streptavidin conjugate (S11237) absorbs maximally at ~434 nm, with bright yellow-green emission ().
• Our blue-fluorescent Alexa Fluor 405 streptavidin (S32351, ) and Pacific Blue streptavidin (S11222, ), yellow-fluorescent Cascade Yellow streptavidin (S11228, ) and orange-fluorescent Pacific Orange streptavidin (S32365, ) absorb maximally between 400 and 410 nm, making them near-perfect matches to the 405 nm spectral line of the violet laser recently developed for fluorescence microscopy and flow cytometry.
• R-phycoerythrin (R-PE) conjugates () of streptavidin (SAPE; S866, S21388) and NeutrAvidin biotin-binding protein (A2660) and the B-phycoerythrin (B-PE) conjugate of streptavidin (S32350) have the most intense fluorescence of all avidin conjugates. Our streptavidin conjugates of R-PE and B-PE have been purified to ensure that all unconjugated streptavidin has been removed (Figure 6.41), making them particularly important labels for multicolor flow cytometry (Section 6.4) and the detection of biotinylated probes on microarrays (Section 8.5, Figure 6.42). Allophycocyanin streptavidin (S868, S32362; ) can be excited by the 633 nm spectral line of the He–Ne laser. In imaging applications, allophycocyanin conjugates are both brighter and more photostable than Cy5 conjugates, with similar spectra (Figure 6.31). Our premium-grade R-PE and allophycocyanin conjugates of streptavidin (S21388, S32362) represent an even further fractionation of our R-PE and allophycocyanin conjugates of streptavidin (S866, S868), respectively.
• We have conjugated R-PE with four of our Alexa Fluor dyes — the Alexa Fluor 610, Alexa Fluor 647, Alexa Fluor 680 and Alexa Fluor 750 dyes — then conjugated these fluorescent proteins to streptavidin to yield labeled conjugates that can be excited with the 488 nm spectral line of the argon-ion laser (Figure 6.34). The long-wavelength emission maxima are 630 nm for the Alexa Fluor 610–R-PE conjugate (S20982), 667 nm for the Alexa Fluor 647–R-PE conjugate (S20992), 702 nm for the Alexa Fluor 680–R-PE conjugate (S20985) and 775 nm for the Alexa Fluor 750–R-PE conjugate (S32363). Emission of the Alexa Fluor 610–R-PE conjugates is shifted to longer wavelengths by about 13 nm relative to that of Texas Red conjugates of R-PE (Figure 6.39). This slightly longer-wavelength emission maximum significantly improves the resolution that can be obtained when using the Alexa Fluor 610–R-PE tandem conjugates in place of Texas Red–R-PE tandem conjugates for multicolor flow cytometry. The Alexa Fluor 647–R-PE tandem conjugates have spectra virtually identical to those of Cy5 conjugates of R-PE but are about three times more fluorescent (Figure 6.38). These tandem conjugates can potentially be used for simultaneous four-color labeling with a single excitation (Figure 6.34). In addition, we have reacted allophycocyanin (APC) with our Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes and then conjugated these labels to streptavidin (S21002, S21005, S21008). The resulting probes can all be excited by the He–Ne laser at 633 nm or krypton-ion laser at 647 nm and have distinguishable emission spectra (Figure 6.37).
• Our DyeMer 488/605, DyeMer 488/615 and DyeMer 488/630 conjugates of streptavidin (S32385, S32386, S32387) are optimized for use in flow cytometry applications. The red-orange–fluorescent DyeMer 488/605, red-fluorescent DyeMer 488/615 and far-red–fluorescent DyeMer 488/630 conjugates are each labeled with a unique bifluorophore comprising two covalently linked fluorophores that act as a donor–acceptor pair for fluorescence resonance energy transfer (FRET). When the green-fluorescent donor dye is excited with the 488 nm spectral line of the argon-ion laser, efficient energy transfer produces fluorescence of the long-wavelength acceptor dye, which emits at 611, 617 or 630 nm (, , ). Any fluorescence from the donor dye due to incomplete FRET can easily be compensated for by setting up compensation circuits to remove unwanted signals. Although their total fluorescence is not as intense as that of the phycobiliprotein tandem conjugates, the DyeMer conjugates exhibit minimal lot-to-lot variation and less interference at the antigen- or biotin-binding site due to the relatively small size of the DyeMer bifluorophores. Moreover, their fluorescence can be excited either at 488 nm or at their longer-wavelength absorption maximum. Because there is some green fluorescence emitted from the donor dye, the DyeMer conjugates were not developed for imaging applications. By carefully choosing bandpass filters that block this green fluorescence or by using a green-fluorescent label for the most abundant target to keep exposure times short, these DyeMer conjugates can be successfully applied to multicolor fluorescence microscopy experiments.

A complete list of our current offerings of fluorophore-, enzyme- and gold-labeled avidins, streptavidins and NeutrAvidin biotin-binding proteins can be