Related Technical Notes
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 avidin–biotin detection 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. In some applications, immobilized avidins are used to capture and release biotinylated targets. In addition to our dye and enzyme conjugates of avidins and streptavidins, this section contains several products that can be used for the affinity isolation of biotin-conjugated molecules and their complexes in cell and tissues.
Our diverse set of biotinylation reagents and biotin conjugates are described in Biotin and Desthiobiotin Conjugates—Section 4.3. Combining one of our biotinylated or DSB-X biotin–labeled secondary 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 sensitive method for the indirect detection of antibodies from various animal sources.
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, 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. We also offer avidin specially packaged in a smaller unit size for extra convenience (A2667). Our avidin, streptavidin and deglycosylated NeutrAvidin biotin-binding protein typically bind greater than 12 µg of biotin per mg protein.
Avidin (A887, A2667; Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9) 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 (S888, Molecular Probe avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9), 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).
We provide an alternative to the commonly used avidin and streptavidin. Our conjugates of NeutrAvidin biotin-binding protein (A2666, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9)—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.
In addition to avidin, streptavidin and NeutrAvidin biotin-binding protein, we offer CaptAvidin biotin-binding protein (C21385, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). 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 7.6.10). 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 separating and purifying biotin conjugates from complex mixtures. Captavidin also provides a regenerable capture reagent for functionalization of surface plasmon resonance (SPR) immunosensors. The biotin-binding capacity of CaptAvidin derivatives is typically at least 10 µg of free biotin per mg protein.
Avidin, streptavidin and NeutrAvidin conjugates are extensively used as secondary detection reagents in histochemical applications (, ), FISH (Figure 7.6.1), flow cytometry, microarrays (Figure 7.6.2) and blot analysis. 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).
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 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 (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, Figure 7.6.1).
- Capture and release. Our unique DSB-X biotin technology (Introduction to Avidin–Biotin and Antibody–Hapten Techniques—Section 4.1) permits the fully reversible labeling of DSB-X biotin derivatives by avidin and streptavidin conjugates. Consequently, targets in cells and tissues or on blots labeled with DSB-X biotin conjugates of antibodies 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; ) 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, and then this preparation is 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 Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7) permit the rapid and quantitative biotinylation of antibodies for combination with avidin–biotin detection methods.
Figure 7.6.1 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 A) using TSA Kit #22 (T20932) with HRP-conjugated streptavidin and Alexa Fluor 488 tyramide or B) using TSA Kit #23 (T20933) with HRP-conjugated streptavidin and Alexa Fluor 546 tyramide. After counterstaining with DAPI (D1306, D3571, D21490), images were obtained using filters appropriate for DAPI, FITC or TRITC.
Figure 7.6.2 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).
Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes often produce substantial background signals when avidin–biotin or streptavidin–biotin detection systems are used to identify cellular targets (). Endogenous biotin is particularly prevalent in mitochondria and in kidney, liver and brain tissues. In mammalian serum and plasma, biotinylated proteins are susceptible to cleavage by endogenous biotinidases, producing free biotin and unlabeled protein.
The reagents in the Endogenous Biotin-Blocking Kit (E21390) can be used to minimize interference from endogenous biotin in these techniques. 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.
Fluorescent avidin and streptavidin are extensively used in DNA hybridization techniques, immunohistochemistry (), MHC tetramer technology (MHC Tetramer Technology—Note 7.2) and multicolor flow cytometry. Our selection of avidin, streptavidin and NeutrAvidin conjugates continues to expand as we introduce new and improved fluorophores and signal amplification technologies (Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). 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 (Figure 7.6.3) and less pH sensitive (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3).
- The orange- and red-orange–fluorescent Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568 and Rhodamine Red-X dyes, and the red-fluorescent Alexa Fluor 594 and Texas Red-X dyes are more fluorescent than traditional Lissamine rhodamine B and Texas Red conjugates, yet have similar excitation and emission maxima.
- The far-red– and infrared-fluorescent Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790 conjugates of streptavidin have fluorescence that is not visible to the eye, but they are efficiently excited by laser light sources and their fluorescence is easily detected with 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, respectively, and their conjugates are more photostable than Cy3 and Cy5 conjugates (Figure 7.6.4).
- The blue-fluorescent Alexa Fluor 350 streptavidin (S11249) displays significantly more fluorescence than AMCA streptavidin (Figure 7.6.5) in side-by-side testing. The blue-fluorescent Alexa Fluor 405 streptavidin (S32351) and Pacific Blue streptavidin (S11222), green-fluorescent Pacific Green streptavidin (S11200), 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 violet laser used for both 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) are significantly brighter than our organic dye–conjugated avidins. Our streptavidin conjugates of R-PE and B-PE have been purified to ensure that all unconjugated streptavidin has been removed (Figure 7.6.6), making them particularly important labels for multicolor flow cytometry (Phycobiliproteins—Section 6.4).
Furthermore, 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—and then conjugated these tandem labels to streptavidin to yield labeled conjugates that can be excited with the 488 nm spectral line of the argon-ion laser. The long-wavelength emission maxima are 628 nm for the Alexa Fluor 610–R-PE conjugate (S20982), 668 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 7.6.7), significantly improving the resolution that can be obtained in 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 7.6.8). We have also conjugated allophycocyanin (APC) with three of our Alexa Fluor dyes—Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 dyes—and then conjugated these tandem 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. 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 avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9.
|Figure 7.6.3 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% formaldehyde, 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 5 minutes, 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 7.6.4 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. The cells were then fixed in 1% formaldehyde, 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 5 minutes, 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 7.6.7 Fluorescence emission spectra of Alexa Fluor 610–R-phycoerythrin streptavidin (S20982, red) and Texas Red dye–R-phycoerythrin streptavidin (blue) tandem conjugates. A) A comparison of the spectra on a relative fluorescence intensity scale for samples prepared with equal absorbance at the excitation wavelength (488 nm). B) The same data normalized to the same peak intensity value to facilitate comparison of the spectral profiles.
Figure 7.6.8 Fluorescence emission spectra of Alexa Fluor 647–R-phycoerythrin streptavidin (S20992; red) and Cy5–R-phycoerythrin streptavidin (blue) tandem conjugates. A) A comparison of the spectra on a relative fluorescence intensity scale for samples prepared with equal absorbance at the excitation wavelength (488 nm). B) The same data normalized to the same peak intensity value to facilitate comparison of the spectral profiles.
We offer 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 (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 immunoassays. They may also be useful as tracers that can be detected with standard enzyme-mediated histochemical methods (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6).
BlockAid blocking solution (B10710) 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 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 other commercially available blocking solutions, 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 with the NIH 3T3, A431, RAW and Jurkat cell lines; however, with the HMC-1 cell line, it did not appear to offer any advantages over standard blocking solutions. 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.
Qdot streptavidin conjugates combine the highly specific binding properties of streptavidin with the exceptional photostability of Qdot nanocrystals (Qdot Nanocrystals—Section 6.6). 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 properly stored archive 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 625 streptavidin conjugate (Q22063, A10196)
- Qdot 655 streptavidin conjugate (Q10121MP)
- Qdot 705 streptavidin conjugate (Q10161MP)
- Qdot 800 streptavidin conjugate (Q10171MP)
- Qdot Streptavidin Sampler Kit (Q10151MP)
Nucleic acid hybridization can be detected by complexation of terminally biotinylated oligonucleotides with Qdot streptavidin conjugates. Qdot streptavidin conjugates in combination with biotinylated antibodies provide increased multiplexing capacity for immunodetection of proteins on both imaging and flow cytometry platforms. Single-particle tracking of molecular motion is a particularly compelling application of Qdot streptavidin conjugates that takes advantage of their extraordinary fluorescence capacity. The principal subjects for these measurements are cell-surface receptors and molecular motors such as myosin. Single-molecule detection uncovers details of molecular motion and assembly that are obscured by the averaging effects of bulk measurements. Qdot nanocrystals are quite essential in such applications, as fluorescence photon output ultimately propagates into the spatial and temporal resolution of the measurements.
Typically, labeling is accomplished using biotinylated ligands such as biotin EGF (E3477, Biotin and Desthiobiotin Conjugates—Section 4.3) and biotin-XX α-bungartoxin (B1196, Biotin and Desthiobiotin Conjugates—Section 4.3) to couple Qdot streptavidin conjugates to the receptor of interest. Because these conjugates incorporate multiple streptavidins (typically 5–10) per Qdot nanocrystal, careful attention must be paid to the streptavidin:ligand stoichiometry to avoid receptor crosslinking mediated by the Qdot streptavidin–biotinyated ligand complex. A molar excess of Qdot streptavidin conjugate over the biotinylated ligand biases the complex stoichiometry towards the desired outcome of one ligand per nanocrystal. Qdot streptavidin conjugates are supplied as 1 µM solutions in 50 mM borate, pH 8.3 containing 0.05% sodium azide and 1 M betaine cryoprotectant in 200 µL units. The Qdot Streptavidin Sampler Kit (Q10151MP) provides 50 µL units of six different Qdot streptavidin conjugates in a single package.
We also offer Qdot ITK streptavidin quantum dots, in which streptavidin is directly attached to the inner amphiphilic coating without a PEG linker. This formulation results in a smaller overall particle size and an increase in the number of streptavidins per nanocrystal relative to PEG-linked conjugates. These characteristics are advantageous in fluorescence resonance energy transfer (FRET) assays for detecting hybridization of target nucleic acid sequences to biotinylated oligonucleotides captured on the Qdot nanocrystal surface. Qdot ITK streptavidin conjugates are described in Qdot Nanocrystals—Section 6.6.
Enzyme conjugates of avidin and streptavidin 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.
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 maximizing retention of both enzyme and carrier protein activity. We have 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). Fluorogenic, chromogenic and chemiluminescent substrates for these enzymes are described in Enzyme Substrates and Assays—Chapter 10. 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.
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 TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2 and Enzyme-Labeled Fluorescence (ELF) Kits in Phosphatase-Based Signal Amplification Techniques—Section 6.3 utilize enzyme conjugates of streptavidin to yield intensely fluorescent staining of cellular targets (Figure 7.6.1, Figure 7.6.9). 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 provides enhanced sensitivity for detection of low-abundance targets.
In collaboration with Nanoprobes, Inc. (www.nanoprobes.com), we offer NANOGOLD and Alexa Fluor FluoroNanogold conjugates of streptavidin to facilitate immunoblotting, light microscopy and electron microscopy applications. 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 therefore provide higher sensitivity. Silver enhancement, such as the system provided in the LI Silver Enhancement Kit (L24919), is described below. Additionally, NANOGOLD particles do not have as high affinity for proteins as do gold colloids, thereby reducing background due to nonspecific binding. Several additional features of NANOGOLD and Alexa Fluor FluoroNanogold conjugates include:
- 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 Fab fragment is nearly 1:1, making this product distinct from the 0.2–10 variable stoichiometry of most colloidal gold–antibody preparations.
NANOGOLD and Alexa Fluor FluoroNanogold products can be used in immunoblotting, light microscopy and electron microscopy. Standard immunostaining methodologies can be used successfully with NANOGOLD and Alexa Fluor FluoroNanogold immunoreagents. Also, because the concentration of antibody and gold is similar to most commercial preparations of colloidal gold antibodies, similar dilutions and blocking agents are appropriate.
We offer several other NANOGOLD and Alexa Fluor FluoroNanogold reagents (NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold conjugates—Table 7.5), including the affinity-purified Fab fragments of the goat anti–mouse IgG and anti–rabbit IgG antibodies covalently conjugated to the 1.4 nm NANOGOLD gold cluster label (Secondary Immunoreagents—Section 7.2). Also available is NANOGOLD monomaleimide (N20345, Thiol-Reactive Probes Excited with Visible Light—Section 2.2), which can be conjugated to thiols in the same way that dyes are conjugated to proteins and nucleic acids.
We offer 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 avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). 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. We prepare 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.
We prepare streptavidin conjugated to 4% beaded crosslinked agarose (S951)—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).
CaptAvidin agarose (C21386) 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 7.6.10). 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.
Figure 7.6.10 Diagram of the use of CaptAvidin agarose (C21386) in affinity chromatography. A biotinylated IgG molecule and target antigen are used as an example.
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.
The high affinity of avidin for biotin was first exploited in histochemical applications in the early 1970s. The use of avidin–biotin techniques has since become widely adopted for diverse detection schemes, although limitations of this method have also been recognized. As an alternative to avidin-based reagents, we offer a high-affinity mouse monoclonal antibody to biotin (03-3700). This anti-biotin antibody can be used to detect biotinylated molecules in immunohistochemistry, in situ hybridization, ELISAs and flow cytometry applications.
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 in 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, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).
For Research Use Only. Not for use in diagnostic procedures.