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

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; Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - 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, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - 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, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - 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, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - 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.

Figure 7.96 Diagram of the use of CaptAvidin agarose in affinity chromatography. A biotinylated IgG molecule and target antigen are used as an example.

Secondary Detection with Avidins

Avidin, streptavidin and NeutrAvidin conjugates are extensively used as secondary detection reagents in histochemical applications (, ), FISH (Detecting Nucleic Acid Hybridization - Section 8.5, Figure 8.92), flow cytometry, microarrays (Detecting Nucleic Acid Hybridization - Section 8.5, Figure 6.42), blot analysis (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - 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 (Polar Tracers - Section 14.3). DSB-X desthiobiocytin and DSB-X biotin ethylenediamine (D20652, D30752; Polar Tracers - Section 14.3) are similar polar tracers that reversibly bind to avidin derivatives.

Figure 8.92 Fluorescence in situ hybridization detected by tyramide signal amplification. Chromosome spreads were prepared from the cultured fibroblast cell line MRC-5 and hybridized with a biotinylated α-satellite probe specific for chromosome 17. The probe was generated by nick translation in the presence of biotinylated dUTP. For detection by TSA, hybridized chromosome spreads were labeled using TSA Kit #22 (T20932) with HRP-conjugated streptavidin and Alexa Fluor 488 tyramide (upper panel) or using TSA Kit #23 (T20933) with HRP-conjugated streptavidin and Alexa Fluor 546 tyramide (lower panel). After counterstaining with DAPI (D1306, D3571, D21490), images were obtained using filters appropriate for DAPI, FITC or TRITC.

Figure 6.42 R-phycoerythrin used to detect DNA on a microarray. A DNA microarray containing a decreasing dilution of calf thymus DNA was hybridized with a biotinylated DNA probe and then incubated with R-phycoerythrin–streptavidin (SAPE; S866, S21388). After washing, the fluorescence signal was detected on a Packard ScanArray 5000 using three different detection configurations: 488 nm excitation (argon-ion laser)/570 nm emission filter (left); 543.5 nm excitation (He–Ne laser)/570 nm emission filter (middle); 543.5 nm excitation (He–Ne laser)/592 nm emission filter (right).

Figure 9.53 Staining glycoproteins and the total protein profile on blots using the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit (P21857). A twofold dilution series of the CandyCane glycoprotein molecular weight standards (C21852) was run an SDS-polyacrylamide gel and blotted onto a PVDF membrane. The blot was first stained with the SYPRO Ruby protein blot stain (S11791) to detect the total protein profile (left). After documentation of the signal, the blot was stained with the Pro-Q Emerald 300 glycoprotein stain (right) provided in the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit.

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 (Tyramide Signal Amplification (TSA) Technology - Section 6.2; Tyramide Signal Amplification (TSA) Kits - Table 6.1; Figure 6.10, Figure 6.11, ) and in several of our kits for ultrasensitive detection of proteins on blots (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, Fluorescence-based Western blot stain kits - 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 (Secondary Immunoreagents - Section 7.2, Molecular Probes' biotinylated and desthiobiotinylated secondary antibodies - 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; Detecting Enzymes That Metabolize Phosphates and Polyphosphates - 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 (Biotin and Desthiobiotin Conjugates - Section 4.3), biotinylated R-phycoerythrin (P811, Phycobiliproteins - Section 6.4), biotinylated FluoSpheres microspheres (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 (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) permit the rapid and quantitative biotinylation of antibodies for combination with avidin–biotin detection methods.

Figure 6.11 Enhancement of estrogen receptor detection sensitivity by tyramide signal amplification (TSA). SKBR3 cells with characteristically low levels of estrogen receptor expression were fixed, permeabilized and treated with H₂O₂ to inhibit endogenous peroxidase activity. A mouse anti–human estrogen receptor monoclonal antibody (Chemicon) was labeled with the Alexa Fluor 488 dye or with biotin using our Zenon Alexa Fluor 488 Mouse IgG₁ Labeling Kit (Z25002) or Zenon Biotin-XX Mouse IgG₁ Labeling Kit (Z25052), respectively. Detection of estrogen receptors using the labeled antibodies was performed on a Becton Dickinson FACScan flow cytometer with excitation at 488 nm. The cellular fluorescence intensity histograms represent detection with Alexa Fluor 488 dye–labeled antibodies (blue), biotinylated antibodies coupled to Alexa Fluor 488 streptavidin (S11223, green) and biotinylated antibodies coupled to HRP–streptavidin and Alexa Fluor 488 tyramide (TSA Kit #22, T20932; orange). The red histogram represents unstained cells.




Figure 7.100 Diagram illustrating the use of streptavidin agarose and a DSB-X biotin bioconjugate in affinity chromatography. A DSB-X biotin–labeled IgG antibody and its target antigen are used as an example.

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

The Image-iT FX Kits (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 (Secondary Immunoreagents - Section 7.2, Fluorescence Characteristics of the Alexa Fluor Dyes in the Image-iT FX Kits - Table 7.7)
• ProLong Gold antifade reagent reduces photobleaching (Figure 23.25, , ; see Fluorescence Microscopy Reference Standards and Antifade Reagents - 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 Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3 for more details)

Figure 23.35 The CultureWell removable chambered coverglass for cell culture (C37000).

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, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23; Alexa Fluor secondary antibodies, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1, Secondary Immunoreagents - Section 7.2; ProLong Gold antifade reagent, P36930, Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1; Image-iT FX signal enhancer, I36933, Secondary Immunoreagents - Section 7.2; CultureWell chambered coverglasses, C37000, C37005, Accessories for Fluorescence Microscopy and Magnetic Separation - 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, Secondary Immunoreagents - 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 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)

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

Fluorescent avidin and streptavidin are extensively used in DNA hybridization techniques, immunohistochemistry (), MHC tetramer technology (MHC Tetramer Technology - Note 7.3 ) 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 (Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - 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 (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3; Figure 7.23, Figure 1.9, , ) (Product Highlight: The Alexa Fluor Dye Series - Note 1.1).
• 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 (Phycobiliproteins - Section 6.4) and the detection of biotinylated probes on microarrays (Detecting Nucleic Acid Hybridization - 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.

Figure 7.23 Comparison of the photostability of immunofluorescent staining by Oregon Green 514 goat anti–mouse IgG antibody (O6383, upper series) and by fluorescein goat anti–mouse IgG antibody (F2761, lower series). Bovine pulmonary arterial endothelial cells were fixed with formaldehyde and permeabilized in cold acetone. Following blocking in 1% BSA, 1% normal goat serum, 0.1% Tween 20 in PBS, samples were incubated for one hour with 60 µg/mL mouse monoclonal anti–human cytochrome oxidase subunit I antibody (A6403, Primary Antibodies for Diverse Applications - Section 7.5), after which they were rinsed and incubated with fluorescent anti–mouse IgG secondary antibodies at 10 µg/mL for 30 minutes. Samples were continuously illuminated and viewed on a fluorescence microscope using an Omega Optical fluorescein longpass filter set, a Star 1 CCD camera (Photometrics) and Image-1 software (Universal Imaging Corp.). Images acquired 0, 20, 40 and 90 seconds after the start of illumination (as indicated in the top left-hand corner of each panel) clearly demonstrate the superior photostability of the Oregon Green 514 conjugate.





Figure 1.9 Photobleaching resistance of the green-fluorescent Alexa Fluor 488, Oregon Green 488 and fluorescein dyes, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 488 (S11223), Oregon Green 488 (S6368) or fluorescein (S869) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). The cells were then fixed in 1% paraformaldehyde, washed and wet-mounted. After mounting, cells were scanned 10 times on a laser-scanning cytometer; laser power levels were 25 mW for the 488 nm spectral line of the argon-ion laser. Scan durations were approximately five minutes apiece, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.




Figure 1.76 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Rhodamine Red-X succinimidyl ester (R6160, ) and Lissamine rhodamine B sulfonyl chloride (L20, L1908, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein. Higher numbers of fluorophores attached per protein are attainable with Rhodamine Red-X dye due to the lesser tendency of this dye to induce protein precipitation.




Figure 1.83 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Texas Red-X succinimidyl ester (T6134, ) and Texas Red sulfonyl chloride (T353, ). Conjugate fluorescence was determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein. Higher numbers of fluorophores attached per protein are attainable with the Texas Red-X dye due to the lesser tendency of this dye to induce protein precipitation.




Figure 1.74 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of 1) fluorescein, 2) rhodamine 6G, 3) tetramethylrhodamine, 4) Lissamine rhodamine B and 5) Texas Red dyes.





Figure 1.24 Absorption spectra of our long-wavelength light–absorbing Alexa Fluor dyes. The Alexa Fluor 635 dye, available conjugated to antibodies, streptavidin and phalloidin, is not included here but its absorption spectrum is very similar to that of the Alexa Fluor 633 dye.




Figure 7.37 Brightness comparison of Molecular Probes' Alexa Fluor 555 goat anti–mouse IgG antibody with Cy3 goat anti–mouse IgG antibody conjugates commercially available from several other companies. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either the Alexa Fluor 555 or Cy3 goat anti–mouse IgG antibody at equal concentrations. Red blood cells were lysed and the samples were analyzed with a flow cytometer equipped with a 488 nm argon-ion laser and a 585 ± 21 nm bandpass emission filter.





Figure 6.31 A comparison of the photobleaching rates of APC and Cy5 conjugates. The microtubules of bovine pulmonary artery endothelial cells were stained with mouse anti–α-tubulin antibody (A11126) in combination with goat anti–mouse IgG labeled antibody with either crosslinked APC (A865, top series) or the Cy5 dye (bottom series). The samples were exposed to continuous illumination, and the images were acquired at 30-second intervals with a Quantex cooled CCD camera (Photometrics) using filter sets appropriate for both APC and Cy5 dye.




Figure 6.34 Normalized fluorescence emission spectra of 1) Alexa Fluor 488 goat anti–mouse IgG antibody (A11001), 2) R-phycoerythrin goat anti–mouse IgG antibody (P852), 3) Alexa Fluor 610–R-phycoerythrin goat anti–mouse IgG antibody (A20980), 4) Alexa Fluor 647–R-phycoerythrin goat anti–mouse IgG antibody (A20990) and 5) Alexa Fluor 680–R-phycoerythrin goat anti–mouse IgG antibody (A20983). The tandem conjugates permit simultaneous multicolor labeling and detection of up to five targets with excitation by a single excitation source — the 488 nm spectral line of the argon-ion laser.



Figure 6.37 Normalized fluorescence emission spectra of 1) allophycocyanin goat anti–mouse IgG antibody (A865), 2) Alexa Fluor 680–allophycocyanin goat anti–mouse IgG antibody (A21000) and 3) Alexa Fluor 750–allophycocyanin goat anti–mouse IgG antibody (A21006). The tandem conjugates permit simultaneous multicolor labeling and detection of up to three targets with excitation by a single excitation source — the 633 nm spectral line of the He–Ne laser.



Figure 6.38 Fluorescence emission spectra of Alexa Fluor 647–R-phycoerythrin streptavidin (S20992; red) and Cy5–R-phycoerythrin streptavidin (Caltag Laboratories; blue) tandem conjugates. Panel A shows a comparison of the spectra on a relative fluorescence intensity scale for samples prepared with equal absorbance at the excitation wavelength (488 nm). Panel B shows the same data normalized to the same peak intensity value to facilitate comparison of the spectral profiles.



Figure 6.39 Fluorescence emission spectra of Alexa Fluor 610–R-phycoerythrin streptavidin (S20982; red) and Texas Red–R-phycoerythrin streptavidin (Caltag Laboratories; blue) tandem conjugates. Panel A shows a comparison of the spectra on a relative fluorescence intensity scale for samples prepared with equal absorbance at the excitation wavelength (488 nm). Panel B shows the same data normalized to the same peak intensity value to facilitate comparison of the spectral profiles.




Figure 6.41 Analytical size-exclusion chromatograms of free streptavidin (S888; red curve, detected by absorption at 280 nm) and R-phycoerythrin streptavidin (SAPE; S866, S21388; blue curve, detected by absorption at 565 nm), demonstrating that the R-phycoerythrin conjugate is substantially free of unconjugated streptavidin.




Figure 6.47 Fluorescence excitation and emission maxima of the FluoSpheres europium luminescent microspheres (F20880, F20881, F20882, F20883, F20884, F20885).




Figure 1.28 Photobleaching resistance of the red-fluorescent Alexa Fluor 647, Alexa Fluor 633, PBXL-3 and Cy5 dyes and the allophycocyanin fluorescent protein, as determined by laser-scanning cytometry. EL4 cells were labeled with biotin-conjugated anti-CD44 antibody and detected by Alexa Fluor 647 (S21374), Alexa Fluor 633 (S21375), PBXL-3, Cy5 or allophycocyanin (APC, S868) streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). The cells were then fixed in 1% paraformaldehyde, washed and wet-mounted. After mounting, cells were scanned eight times on a laser-scanning cytometer; laser power levels were 18 mW for the 633 nm spectral line of the He–Ne laser. Scan durations were approximately five minutes apiece, and each repetition was started immediately after completion of the previous scan. Data are expressed as percentages derived from the mean fluorescence intensity (MFI) of each scan divided by the MFI of the first scan. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National Cancer Institute.

Figure 1.71 Effect of protein conjugation on the absorption spectrum of tetramethylrhodamine. The absorption spectrum of tetramethylrhodamine conjugated to goat anti–mouse IgG antibody (TMR-GAM, T2762) shows an additional peak at about 520 nm when compared with the spectrum of the same concentration of the free dye (TMR). Partial unfolding of the protein in the presence of 4.8 M guanidine hydrochloride (TMR-GAM + GuHCl) results in a spectrum more similar to that of the free dye.

Figure 7.31 Comparison of the relative fluorescence of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) streptavidin () and Alexa Fluor 350 streptavidin, a sulfonated AMCA derivative (S11249, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein.

A complete list of our current offerings of fluorophore-, enzyme- and gold-labeled avidins, streptavidins and NeutrAvidin biotin-binding proteins can be found in Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23. To obtain the maximal fluorescence signal from some conjugates, free D-biotin (B1595, B20656) can be added (Add Free Biotin to Obtain Brighter Signals from Some Fluorescent Avidin Conjugates - Note 7.4).

Streptavidin-, NeutrAvidin- and Biotin-Labeled Fluorescent Microspheres

Molecular Probes offers streptavidin, NeutrAvidin and biotin conjugates of the intensely fluorescent FluoSpheres and TransFluoSpheres polystyrene microspheres in a variety of colors and sizes, including our europium and platinum luminescent beads labeled with the NeutrAvidin biotin-binding protein for time-resolved fluorometry (Figure 6.47, Figure 6.48; Molecular Probes' europium and platinum luminescent FluoSpheres microspheres - Table 14.8). Because single fluorescent microspheres can be detected, FluoSpheres and TransFluoSpheres beads have significant potential for ultrasensitive flow cytometry applications and immunodiagnostic assays. They may also be useful as tracers that can be detected with standard enzyme-mediated histochemical methods (FluoSpheres and TransFluoSpheres Microspheres for Tracing - Section 14.6).

BlockAid blocking solution (B10710, BlockAid Blocking Solution) is a protein-based reagent designed principally for use with our streptavidin-, NeutrAvidin- and biotin-labeled FluoSpheres (Summary of biotin-, streptavidin- and NeutrAvidin biotin-binding protein-labeled FluoSpheres microspheres - Table 6.8) and our streptavidin- and NeutrAvidin-labeled TransFluoSpheres microspheres (Summary of Molecular Probes' TransFluoSpheres fluorescent microspheres - Table 6.9). Protein- and other macromolecule-labeled microspheres have hydrophobic regions that may cause them to bind to nontarget surfaces in some experimental systems. Although this nonspecific binding can often be relieved by the use of a blocking solution, we have found that microspheres require a stronger blocking solution than those in common use. In our tests, the BlockAid blocking solution was mixed with streptavidin-labeled FluoSpheres microspheres, which were then used to stain several different cell types for subsequent analysis by flow cytometry. We found the BlockAid blocking solution to be superior to blocking solutions available from other companies, as well as to several standard blocking solutions described in the scientific literature for reducing nonspecific binding of labeled microspheres. BlockAid blocking solution has been found to be effective in flow cytometry applications involving NIH 3T3, A431, RAW and Jurkat cell lines. We expect that the BlockAid blocking solution will be useful for reducing the nonspecific binding of protein-coated or other macromolecule-coated microspheres in a variety of flow cytometry and microscopy applications. It may also be useful as a general blocking agent in a variety of other assays.

NANOGOLD and Alexa Fluor FluoroNanogold Streptavidin

In collaboration with Nanoprobes, Inc.(http://www.nanoprobes.com/), Molecular Probes offers NANOGOLD and Alexa Fluor FluoroNanogold conjugates of streptavidin to facilitate immunoblotting, light microscopy and electron microscopy applications (NANOGOLD(R) and FluoroNanogold Conjugates). NANOGOLD conjugates are covalently conjugated to the 1.4 nm NANOGOLD gold cluster label, whereas Alexa Fluor FluoroNanogold conjugates are coupled to both a NANOGOLD label and either the Alexa Fluor 488 or Alexa Fluor 594 fluorophore, resulting in gold clusters with green or red fluorescence, respectively. Alexa Fluor FluoroNanogold streptavidin conjugates have all the advantages of the NANOGOLD conjugates, with the additional benefit that they may be used for correlative fluorescence, light and electron microscopy ().

NANOGOLD gold clusters have several advantages over colloidal gold. They develop better with silver than do most gold colloids and, as a result, provide higher sensitivity. Additionally, NANOGOLD particles do not have as high affinity for proteins as do gold colloids, thereby reducing any background due to nonspecific binding. Several additional advantages of NANOGOLD and Alexa Fluor FluoroNanogold streptavidin over colloidal gold conjugates include:

• The NANOGOLD gold clusters are an extremely uniform (1.4 nm ± 10% diameter) and stable compound, not a gold colloid.
• NANOGOLD gold clusters are smaller than a complete IgG (H+L) antibody — approximately 1/15 the size of an Fab fragment — and therefore will be able to better penetrate cells and tissues, reaching antigens that are inaccessible to conjugates of larger gold particles.
• NANOGOLD conjugates contain absolutely no aggregates, as they are chromatographically purified through gel filtration columns. This feature is in sharp contrast to colloidal gold conjugates, which are usually prepared by centrifugation to remove the largest aggregates and frequently contain significantly smaller aggregates.
• The ratio of NANOGOLD particle to streptavidin is nearly 1:1, making this product distinct from the 0.2–10 variable stoichiometry of most colloidal gold preparations.
• NANOGOLD cluster–stained targets develop better with silver than do most gold colloids, resulting in higher sensitivity. Silver enhancement, such as the system provided in the LI Silver Enhancement Kit (L24919, Figure 7.54), can be used for light microscopy and immunoblotting to provide improved results; see Secondary Immunoreagents - Section 7.2 for a complete description.

The sensitivity of NANOGOLD streptavidin, and presumably Alexa Fluor FluoroNanogold streptavidin, in immunohistochemical applications can be further improved by first using one of our Tyramide Signal Amplification (TSA) Kits containing biotin tyramide (Tyramide Signal Amplification (TSA) Technology - Section 6.2, Tyramide Signal Amplification (TSA) Kits - Table 6.1) and then detecting the biotin tyramide conjugates with NANOGOLD streptavidin, enhanced by silver staining.

Molecular Probes offers several other NANOGOLD and Alexa Fluor FluoroNanogold reagents (NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold conjugates - Table 7.10), including the affinity-purified Fab fragments of the goat anti–mouse IgG, goat anti–rabbit IgG and rabbit anti–goat IgG antibodies covalently conjugated to the 1.4 nm NANOGOLD gold cluster label (Secondary Immunoreagents - Section 7.2). Also available are NANOGOLD mono(sulfosuccinimidyl ester) (N20130, Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers - Section 1.6, Figure 1.89) and NANOGOLD monomaleimide (N20345, Thiol-Reactive Probes Excited with Visible Light - Section 2.2, Figure 2.22), which can be conjugated to amines and thiols, respectively, in the same way that dyes are conjugated to proteins and nucleic acids.

Figure 1.89 Reaction of NANOGOLD mono(sulfosuccinimidyl ester) (N20130) with a primary amine. Image courtesy of Nanoprobes, Inc.



Figure 2.22 Reaction of NANOGOLD monomaleimide (N20345) with a thiol. Image courtesy of Nanoprobes, Inc.

Colloidal Gold Complexes

Molecular Probes offers Alexa Fluor 488 dye–labeled colloidal gold conjugates, including those of goat anti–mouse IgG (A31560, A31561; Secondary Immunoreagents - Section 7.2) and goat anti–rabbit IgG antibodies (A31565, A31566; Secondary Immunoreagents - Section 7.2) and streptavidin (A32360, A32361; Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23). These conjugates, which have been adsorbed to 5 nm or 10 nm gold colloids, may be used as probes in immunoblotting, light microscopy, fluorescence microscopy or electron microscopy. The fluorescence of these conjugates can be easily detected by standard techniques, but visualization of colloidal gold can be greatly improved using silver-enhancement methods, such as those we provide in the LI Silver Enhancement Kit (L24919) described in Secondary Immunoreagents - Section 7.2.

Combining fluorescent secondary detection reagents with colloidal gold to form functional complexes is difficult because the fluorescence of fluorophores such as fluorescein is significantly quenched by proximity to the colloidal gold. Molecular Probes makes fluorescent colloidal gold complexes with our Alexa Fluor 488 dye, a dye that has superior brightness and photostability. Our Alexa Fluor 488 dye–labeled colloidal gold complexes of anti-IgG antibody and of streptavidin can potentially be used to perform correlated immunofluorescence and electron microscopy in a two-step labeling procedure, rather than in the three-step indirect labeling procedure that is required with conventional nonfluorescent colloidal gold complexes of anti-IgG antibodies or streptavidin.

Enzyme conjugates are extensively used in enzyme-linked immunosorbent assays (ELISAs), blotting techniques, in situ hybridization and cytochemistry and histochemistry. Enzyme-mediated in situ techniques using these conjugates provide better resolution and are safer, more sensitive and faster than radioactive methods. Most frequently, the enzymes of choice are horseradish peroxidase, alkaline phosphatase and Escherichia coli β-galactosidase because of their high turnover rate, stability, ease of conjugation and relatively low cost. Molecular Probes has prepared highly active streptavidin and NeutrAvidin biotin-binding protein conjugates of horseradish peroxidase (S911, A2664), alkaline phosphatase (S921), β-galactosidase (S931) and β-lactamase TEM-1 (S31569), as well as biotin-XX horseradish peroxidase (P917, Enzyme Conjugates). Fluorogenic substrates for ELISAs are often much more sensitive than chromogenic substrates in these important assays. Our fluorogenic, chromogenic and chemiluminescent substrates for these assays are described in Enzyme Substrates - Chapter 10.

Our enzyme conjugates of streptavidin and NeutrAvidin biotin-binding protein are prepared by techniques that yield an approximate 1:1 ratio of enzyme to avidin analog, thus ensuring maximum retention of activity of both enzyme and carrier protein. We offer streptavidin conjugates of alkaline phosphatase, horseradish peroxidase, β-galactosidase and β-lactamase (S921, S911, S931, S31569) and the NeutrAvidin conjugate of horseradish peroxidase (A2664). To decrease background problems, researchers often prefer to use the biotin-XX conjugate of horseradish peroxidase (P917) in conjunction with an avidin or streptavidin bridge for indirect detection of a wide array of biotinylated biomolecules. Our biotinylated horseradish peroxidase conjugate is prepared with a reactive biotin-XX derivative, which contains the longest available spacer and allows high avidin affinity.

A principal application of HRP and alkaline phosphatase conjugates of avidins and secondary antibodies is in enzyme-amplified histochemical staining of cells and tissues. Several of the Tyramide Signal Amplification (TSA) Kits (Tyramide Signal Amplification (TSA) Kits - Table 6.1) in Tyramide Signal Amplification (TSA) Technology - Section 6.2 and Enzyme-Labeled Fluorescence (ELF) Kits in Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3 utilize enzyme conjugates of streptavidin to yield intensely fluorescent staining of cellular targets (Figure 6.10, Figure 6.11, , ). These kits are very useful for immunofluorescence, in situ hybridization and flow cytometry. Use of a combination of the TSA and ELF technologies or double application of TSA methods promises to provide the highest sensitivity known for detection of low-abundance targets.

Figure 6.10 Detection of epidermal growth factor (EGF) receptors directly or with signal amplification. Cells expressing high (A431 cells, panel A) and low (NIH 3T3 cells, panel B) levels of EGF receptors were either directly labeled with the preformed Alexa Fluor 488 complex of biotinylated epidermal growth factor (E13345, blue) or indirectly labeled with biotinylated EGF (E3477) followed by either Alexa Fluor 488 streptavidin (S11223, green) or HRP-conjugated streptavidin and Alexa Fluor 488 tyramide (purple), components of our TSA Kit #22 (T20932).

The use of horseradish peroxidase (HRP) for enzyme-amplified immunodetection, commonly referred to as immunoperoxidase labeling, is a well-established standard histochemical technique. The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse IgG primary antibodies (D22185) and biotinylated antibodies and tracers (D22187). Each kit contains:

• Diaminobenzidine (DAB)
• HRP-labeled goat anti–mouse IgG antibody (in Kit D22185) or streptavidin (in Kit D22187) conjugate
• H₂O₂ reaction additive
• Blocking reagent
• Staining buffer
• A detailed staining protocol (Diaminobenzidine Histochemistry Kits)

Each kit provides sufficient materials to stain approximately 200 slides.

Streptavidin Agarose

Molecular Probes prepares streptavidin conjugated to 4% beaded crosslinked agarose (S951, Agarose and Acrylamide Conjugates) — a matrix that can be used to isolate biotinylated peptides, proteins, hybridization probes, haptens and other molecules. In addition, biotinylated antibodies can be bound to streptavidin agarose to generate an affinity matrix for the large-scale isolation of antigens. For instance, staurosporine-treated myotubules have been incubated with biotinylated α-bungarotoxin (B1196, Probes for Neurotransmitter Receptors - Section 16.2) in order to isolate the acetylcholine receptors (AChRs) on streptavidin agarose and assess staurosporine's effect on the degree of phosphorylation of this receptor. Streptavidin agarose has also been used to investigate the turnover of cell-surface proteins that had previously been derivatized with an amine-reactive biotin (B1582, Biotinylation and Haptenylation Reagents - Section 4.2).

DSB-X Bioconjugate Isolation Kit #1

The DSB-X Bioconjugate Isolation Kit #1 (D20658) uses our unique DSB-X biotin technology for the easy affinity isolation of DSB-X biotin–labeled bioconjugates under extremely gentle conditions. DSB-X biotin is a derivative of desthiobiotin (Figure 4.1), a stable biotin precursor. DSB-X biotin utilizes a seven-atom spacer to increase the ability of the DSB-X biotin conjugate to bind in the deep biotin-binding pocket of streptavidin or avidin. Whereas harsh chaotropic agents and low pH (6.0 M guanidine HCl, pH 1.5) are required to dissociate a biotin complex from avidin or streptavidin, streptavidin agarose has only a moderate affinity for conjugates of DSB-X biotin. Therefore, binding can be rapidly reversed by adding excess D-desthiobiotin (D20657) or natural D-biotin (B1595, B20656) to the matrix at neutral pH and at room temperature (or below).

Figure 4.1 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).

Once bound to the streptavidin agarose matrix, the bioconjugate — an antibody, enzyme, oligonucleotide, nucleic acid, drug or other DSB-X biotin conjugate — can bind to its target, which may be from a variety of sources, including cell or tissue extracts (Figure 7.100). Gentle elution of the entire complex allows subsequent analysis of the affinity-isolated product by electrophoresis or other means. Elution with D-desthiobiotin rather than D-biotin may permit reuse of the matrix.

The DSB-X Bioconjugate Isolation Kit #1 (D20658) contains:

• Streptavidin agarose (5 mL of a sedimented bead suspension)
  
• Solutions of D-desthiobiotin and D-biotin
  
• Purification columns
• A suggested protocol for binding and release of DSB-X biotin conjugates (DSB-X Bioconjugate Isolation Kit #1, with streptavidin agarose)

Molecular Probes provides a variety of antibody conjugates of DSB-X biotin (Secondary Immunoreagents - Section 7.2, Molecular Probes' biotinylated and desthiobiotinylated secondary antibodies - Table 7.11) as well as DSB-X biotin hydrazide (D20653, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) for selective labeling and capture of periodate-oxidized glycoproteins and polysaccharides. Labeling of amine residues of other proteins and other biomolecules is easily accomplished with the reagents in our DSB-X Biotin Protein Labeling Kit (D20655, Kits for Labeling Proteins and Nucleic Acids - Section 1.2).

CaptAvidin Agarose

CaptAvidin agarose (C21386, CaptAvidin Biotin-Binding Protein) is another versatile form of a biotin-binding protein in that its affinity for biotinylated molecules can be completely reversed by raising the pH to 10, permitting the facile separation and isolation of biotin-labeled molecules from complex mixtures (Figure 7.96). This form of agarose-immobilized biotin-binding protein has been used to purify immunoglobulin from whole rabbit serum and to isolate anti-transferrin antibodies directly from rabbit antiserum.

Captivate Ferrofluid Streptavidin

Captivate ferrofluid streptavidin (C21476, Captivate Ferrofluid Conjugates and Related Products) is a versatile product for rapid separation of biotinylated and DSB-X biotin–conjugated biomolecules and their targets from complex mixtures, including those in cell and tissue extracts and bodily fluids. Combination of Captivate ferrofluid streptavidin with DSB-X biotin (Figure 4.1) technology enables the selective capture and release of rare populations of viable cells by DSB-X biotin–conjugated antibodies to cell surface markers (Figure 7.104). A potentially important application of this technique is the detection of specific protein–protein and protein–nucleic acid interactions through selective isolation and release of their intact complexes.

The Captivate ferrofluid products are unique in that they represent the only superparamagnetic particles available that allow both cell sorting and cell-based imaging to be performed simultaneously by use of the Captivate microscope-mounted magnetic yoke assembly and associated Captivate disposable sample chambers (C24700, C24701; Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3; Figure 7.55). The Captivate microscope-mounted magnetic yoke assembly includes one free set of 10 disposable sample chambers. Use of Captivate ferrofluid streptavidin in combination with biotin- or DSB-X biotin–conjugated probes permits the simultaneous isolation, visualization and counting of cells that are targets of the antibody by any researcher with access to a standard low-cost microscope with a 10× objective (). Also, when used to capture DSB-X biotin–labeled antibodies to cell-surface antigens, the Captivate ferrofluid can be completely separated from the labeled cells by incubation with D-biotin (B1595, B20656; Biotinylation and Haptenylation Reagents - Section 4.2) or D-desthiobiotin (D20657, Biotinylation and Haptenylation Reagents - Section 4.2). In addition, the Captivate ferrofluid products should have advantages over other commercially available magnetic particles in liquid-handling robotic systems.

Molecular Probes also has available Captivate magnetic separators for both microplates (C24702, Figure) and microtubes (C24703, Figure) that we find to be particularly useful with the Captivate ferrofluid products. The microplate separator is compatible with most 96-well microplates, whereas the microtube separator can accommodate six 1.5 mL microcentrifuge tubes. Both separators provide excellent separation efficiency and pull magnetic particles to one side, allowing easier removal of supernatants.

DSB-X Bioconjugate Isolation Kit #2

The DSB-X Bioconjugate Isolation Kit #2 (D20659) utilizes DSB-X biotin agarose for affinity isolation of any avidin or streptavidin conjugate (Figure 7.106). DSB-X biotin agarose links desthiobiotin to agarose through a seven-atom spacer (Figure 4.1). Binding of the avidin or streptavidin conjugate can be fully reversed under extremely gentle conditions by addition of D-biotin or desthiobiotin. We have shown that when desthiobiotin (but not D-biotin) is used to reverse the binding, the avidin or streptavidin biotin-binding sites are fully saturated by the desthiobiotin; however, it is not necessary to remove the desthiobiotin before use of the affinity-purified avidin or streptavidin conjugate to label a biotin-conjugated probe. The reagents in this kit can be used to isolate streptavidin conjugates that are free from enzymes or other biomolecules when forming protein–protein or protein–nucleic acid conjugates. The DSB-X Bioconjugate Isolation Kit #2 (D20659) contains:

• DSB-X biotin agarose (5 mL of a sedimented bead suspension)
• Solutions of D-desthiobiotin and D-biotin
• Purification columns
• Suggested protocol for binding and release of DSB-X biotin conjugates (DSB-X Bioconjugate Isolation Kit #2, with DSB-X biotin agarose)

The DSB-X biotin agarose in this kit can potentially be reused several times.

Cell-Surface Biotinylation Kit

Biotin-XX sulfosuccinimidyl ester is a membrane-impermeant, amine-reactive compound that may be used to label proteins exposed on the surface of live cells (Figure 4.5). The sulfosuccinimidyl ester forms an extremely stable conjugate with cell-surface proteins, and the biotin provides a convenient hapten for subsequent isolation or analysis with an avidin-based protein such as streptavidin, NeutrAvidin or CaptAvidin biotin-binding protein or the Captivate ferrofluid streptavidin conjugate (C21476, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). Cell-surface biotinylation techniques have been employed to differentially label proteins in the apical and basolateral plasma membranes of epithelial cells. These techniques are also well suited for studying internalization of membrane proteins and cell-surface targeting of proteins. The

FluoReporter Cell-Surface Biotinylation Kit (F20650) provides a convenient method to label proteins exposed on the cell surface including, but not limited to, membrane proteins. This kit includes:

• Biotin-XX sulfosuccinimidyl ester
• Anhydrous DMSO for preparing stable stock solutions
• A detailed protocol for cell-surface biotinylation (FluoReporter(R) Cell-Surface Biotinylation Kit)

The supplied protocol for cell-surface biotinylation is easy to perform and can be completed in less than one hour. Biotinylated proteins can be subsequently identified using the reagents in some of our Pro-Q, Amplex Gold and DyeChrome Western Blot Kits (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4).

Acrylamide Conjugates for Immobilization of Avidins in Polymers

Streptavidin acrylamide (S21379), which is prepared from the succinimidyl ester of 6-((N-acryloyl)amino)hexanoic acid (acryloyl-X, SE; A20770, Chemical Crosslinking Reagents - Section 5.2), is a reagent that may be useful for preparing biosensors. A similar streptavidin acrylamide has been shown to copolymerize with acrylamide on a polymeric surface to create a uniform monolayer of the immobilized protein. The streptavidin can then bind biotinylated ligands, including biotinylated hybridization probes, enzymes, antibodies and drugs. CaptAvidin acrylamide (C21387) is expected to have similar utility, but offers an advantage — the bond that it forms with biotinylated probes can be reversed at about pH 10.

In addition to the direct conjugates of avidins, Molecular Probes offers an extensive selection of biotinylated products for use in conjunction with avidins; see Biotin Derivatives and Haptens - Chapter 4 for a complete list of our biotinylation reagents and biotin conjugates. Molecular Probes offers a broad selection of biotinylating reagents, including FluoReporter Biotin-XX and Biotin/DNP Protein Labeling Kits (F2610, F6347, F6348; Kits for Labeling Proteins and Nucleic Acids - Section 1.2). Reactive forms of DSB-X biotin and our unique DSB-X biotin bioconjugates are also described in Biotin Derivatives and Haptens - Chapter 4.

Our diverse set of biotin and DSB-X biotin conjugates is described in Biotin and Desthiobiotin Conjugates - Section 4.3. Combining one of our biotinylated or DSB-X biotin–labeled antibodies (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1) with a fluorescent dye– or enzyme-labeled avidin provides an easy method for indirect detection of antibodies from various animal sources. Biotinylated R-phycoerythrin (P811, Phycobiliproteins - Section 6.4) can be used with an avidin or streptavidin bridge to detect biotinylated biomolecules; this bridging technique may substantially reduce the nonspecific staining that is commonly seen when using phycobiliproteins for immunohistochemical applications.

Biotinylated liposomes can be prepared using Molecular Probes' biotin conjugates of phosphoethanolamine (B1550, B1616; Biotin and Desthiobiotin Conjugates - Section 4.3). Avidin has been used to form a bridge between a biotinylated liposome loaded with fluorescent dyes and a target-specific biotinylated detection reagent. Biotinylated liposomes containing carboxyfluorescein have been employed in an immunoassay that was reported to be both faster and 100 times more sensitive than the comparable peroxidase-based ELISA.

As an alternative to avidin-based reagents, Molecular Probes offers unlabeled, Alexa Fluor 488 dye–labeled and Alexa Fluor 594 dye–labeled versions of a high-affinity mouse monoclonal antibody to biotin (A11242, A31801, A31800). Our anti-biotin antibody can be used to detect biotinylated molecules in immunohistochemistry, in situ hybridization, ELISAs and Western blot applications (Labeled and Unlabeled Anti-Biotin). Somewhat unexpectedly, our anti-biotin antibody retains high affinity for desthiobiotin; consequently, its binding to DSB-X biotin bioconjugates cannot be easily reversed by addition of free D-biotin.

It has been shown that certain monoclonal antibodies to biotin have biotin-binding motifs that are similar to those seen for avidin and streptavidin. Anti-biotin antibody has been shown to selectively stain endogenous biotin-dependent carboxylase proteins used in fatty acid synthesis of the mitochondria. Nonspecific staining of mitochondrial proteins by labeled avidins and by anti-biotin antibodies can be a complicating factor when using avidin–biotin techniques (). This nonspecific binding can usually be blocked by pretreatment of the sample with the reagents in our Endogenous Biotin-Blocking Kit (E21390, see above).

Especially useful for indirect immunofluorescence, the green-fluorescent Alexa Fluor 488 conjugate exhibits excitation/emission maxima similar to fluorescein, but its fluorescence is brighter, more photostable and pH insensitive. Likewise, the red-fluorescent Alexa Fluor 594 conjugate is spectrally similar to but much more fluorescent than Texas Red conjugates, making it particularly useful for multilabeling experiments in combination with green-fluorescent probes. Because it is a mouse IgG₁ antibody, our anti-biotin antibody can be used with any of our Zenon Mouse IgG₁ Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) to create even more alternatives to avidin-based reagents for the detection of biotinylated probes.