Unlike the polydisperse dextrans (Fluorescent and Biotinylated Dextrans—Section 14.5), fluorescent protein tracers have molecular weights that are reasonably well defined (bovine serum albumin (BSA) ~66,000 daltons; ovalbumin ~45,000; codfish parvalbumin 12,328 daltons, casein ~23,600 daltons; monomeric subunit B of cholera toxin ~12,000 daltons; horseradish peroxidase (HRP) ~40,000 daltons; soybean trypsin inhibitor ~21,500 daltons; Phaseolus vulgaris leucoagglutinin (PHA-L) ~126,000 daltons). Some of their applications are similar to those of dextran tracers, although protein conjugates may be more susceptible to proteolysis. Through creative modifications of the proteins, however, researchers have been able to use these conjugates to target receptors, to detect protease activity and to measure intracellular pH.
Fluorescent BSA, Ovalbumin and Parvalbumin
For use as protein tracers, we supply bovine serum albumin (BSA) fluorescently labeled with:
In addition to these BSA conjugates, we prepare fluorescein and Texas Red conjugates of ovalbumin (O23020, O23021), as well as the intensely fluorescent Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 and Alexa Fluor 647 conjugates (O34781, O34782, O34783, O34784). Ovalbumin is a protein with a relatively low molecular weight (~45,000 daltons) that is primarily useful as an antigen for activation of dendritic cells, macrophages and B cells.
We also offer the Alexa Fluor 488 conjugate of codfish parvalbumin (P23012), which is the smallest fluorescent protein tracer in this section. Parvalbumin, a stable and protease-resistant protein with a molecular weight of 12,328 daltons, is found in the skeletal muscle of vertebrates and the endocrine glands of mammals.
Dinitrophenylated Bovine Serum Albumin
We offer BSA that has been conjugated to multiple dinitrophenyl hapten molecules (DNP-BSA, A23018), a reagent that is commonly used to study Fc receptor–mediated immune function and the IgE- and IgG-mediated responses to crosslinking of DNP-specific antibodies. Our dinitrophenylated BSA typically has ~25 dinitrophenyl substituents per molecule of BSA. For detection of DNP-BSA, we offer unlabeled rabbit anti-DNP antibody prepared against DNP-conjugated keyhole limpet hemocyanin (anti–DNP-KLH antibody) and the Alexa Fluor 488 dye, fluorescein and biotin-XX antibody conjugates (A6430, A11097, A6423, A6435; Anti-Dye and Anti-Hapten Antibodies—Section 7.4), as well as a Qdot 555 rat monoclonal anti-dinitrophenol antibody (Q17421MP).
DQ BSA, DQ Ovalbumin and DQ Collagen
DQ substrates, including the DQ Green BSA and DQ Red BSA (D12050, D12051) and DQ ovalbumin (D12053) are based on the strong fluorescence quenching effect observed when proteins are heavily conjugated with BODIPY dyes or, in the case of DQ collagen conjugates (D12052, D12060), with multiple fluorescein dyes. Upon hydrolysis of the proteins to single dye–labeled peptides by proteases, this quenching is relieved (Figure 14.7.1). This technology provides an easy continuous assay for numerous proteases (Detecting Peptidases and Proteases—Section 10.4). When complexed with an anti-BSA rabbit IgG antibody (A11133), the DQ BSA products are phagocytosed into live cells via the Fc receptor (Probes for Following Receptor Binding and Phagocytosis—Section 16.1, Figure 14.7.2), where they are broken down into fluorescent peptides (Figure 14.7.1). DQ Green BSA, which gives bright green fluorescence upon proteolysis, and DQ Red BSA, which yields red-fluorescent hydrolysis products, are packaged in sets of five vials, each containing 1 mg of the substrate. Our Fc OxyBURST Green assay reagent (F2902, Probes for Following Receptor Binding and Phagocytosis—Section 16.1), which is based on a similar concept, comprises BSA that has been conjugated to a dihydrofluorescein derivative and then complexed with anti-BSA IgG. Upon binding to the Fc receptor, the immune complex is internalized and oxidized to a fluorescent product within the phagovacuole.
We have found our highly quenched DQ ovalbumin (D12053) to be particularly useful for antigen processing and presentation studies. Ovalbumin conjugates are internalized via the mannose receptor–mediated endocytosis pathway and are recognized by antigen-presenting cells. BSA that was heavily labeled with fluorescein (FITC) has been used for these studies. However, fluorescence of the intracellular hydrolysis products of FITC BSA is significantly decreased by the acidity of the phagovacuole. In contrast, the bright fluorescence of the BODIPY FL fluorophore in DQ ovalbumin is completely independent of the pH. The fluorescence generated after DQ collagen (D12052) is hydrolyzed by cellular collagenase has been used to visualize the migratory pathway followed by tumor cells during invasion of a gelatin matrix and to image proteolysis by living breast cancer cells.
Figure 14.7.1 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in our EnzChek Protease Assay Kits (E6638, E6639), EnzChek Ultra Amylase Assay Kit (E33651), EnzChek Gelatinase/Collagenase Assay Kit (E12055), EnzChek Elastase Kit (E12056), EnzChek Lysozyme Assay Kit (E22013)—as well as the stand-alone quenched substrates DQ BSA (D12050, D12051), DQ collagen (D12052, D12060), DQ ovalbumin (D12053) and DQ gelatin (D12054)—relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.
Figure 14.7.2 Immune complex of DQ BSA conjugate (D12050, D12051) with an anti–bovine serum albumin (BSA) antibody (A11133) for the fluorescent detection of the Fc receptor–mediated phagocytosis pathway. The DQ BSA is a derivative of BSA that is labeled to such a high degree with either the green-fluorescent BODIPY FL or red-fluorescent BODIPY TR-X dye that the fluorescence is self-quenched. Upon binding to an Fc receptor, the nonfluorescent immune complex is internalized and the protein is subsequently hydrolyzed to fluorescent peptides within the phagovacuole.
Injectable Contrast Reagents for Small Animal in vivo Imaging Applications
The study of vascularization—often in tumors—requires a probe that will adequately distribute throughout the system under study without being cleared from the tissue prior to the time needed to perform imaging. For this purpose, bovine serum albumin (BSA) and human serum transferrin labeled with near-infrared–fluorescent Alexa Fluor 680 (S34788, S34790) and Alexa Fluor 750 (S34789, S34791) dyes are offered as injectable contrast agents in small animal in vivo imaging. These SAIVI injectable contrast agents have been optimized for emission intensity and tested as reagents for the in vivo imaging of regions of blood pooling and vascularization in an inflammatory disease model in mice. Infrared fluorescent microspheres (Microspheres—Section 6.5) can also be used in a similar manner.
BODIPY FL casein and BODIPY TR-X casein, which are components of our EnzChek Protease Assay Kits (E6638, E6639), are substrates for metallo-, serine, acid and sulfhydryl proteases, including cathepsin, chymotrypsin, elastase, papain, pepsin, thermolysin and trypsin (Detecting Peptidases and Proteases—Section 10.4). These casein-based substrates are heavily labeled and therefore highly quenched conjugates; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Protease-catalyzed hydrolysis relieves this quenching, yielding brightly fluorescent peptides (Figure 14.7.1).
Each kit provides sufficient reagents for ~100 assays using 2 mL assay volumes and standard fluorescence cuvettes or ~1000 assays using 200 µL assay volumes and 96-well microplates.
The EnzChek Protease Assay Kits, as well as the DQ BSA and DQ ovalbumin substrates mentioned above, have significant potential as:
- Indicators of protease contamination in culture media
- Fluorogenic substrates for circulating or secreted proteases in extracellular fluids
- Nontoxic, pH-insensitive markers for phagocytic cells, which will ingest and eventually cleave the quenched casein substrates to yield fluorescent BODIPY FL– or BODIPY TR-X–labeled peptides
- Microinjectable tracers to detect enhanced protease activity associated with cell activation and fusion
- Nontoxic markers for assessing various cell-loading and cell-transfection techniques, including electroporation (Techniques for loading molecules into the cytoplasm—Table 14.1)
The peptide hydrolysis products of BODIPY FL casein exhibit green fluorescence that is optimally excited by the argon-ion laser at 488 nm, permitting flow sorting of the cells. The red-fluorescent BODIPY TR-X–labeled peptides, with excitation and emission spectra similar to those of the Texas Red fluorophore, should be useful for multilabeling experiments or measurements in the presence of green autofluorescence. Following intracellular hydrolysis, some of the lower molecular weight fluorescent peptides may diffuse into other organelles or pass thorough gap junctions.
Our Alexa Fluor 488, Alexa Fluor 594 and Alexa Fluor 647 conjugates (L11270, L32456, L32457) of Phaseolus vulgaris leucoagglutinin (PHA-L) are excellent fluorescent anterograde tracers. Iontophoretically injected PHA-L clearly demonstrates the morphological features of the filled neurons at the injection site and the labeled axons and axon terminals. Furthermore, PHA-L that has been transported is not degraded over long periods. PHA-L has been used in combination with our biotin dextran amines (BDA dextrans, Fluorescent and Biotinylated Dextrans—Section 14.5) or the fluorescent dextrans fluoro-ruby and mini-ruby (D1817, D3312; Fluorescent and Biotinylated Dextrans—Section 14.5) to demonstrate their similar transport properties and to show the distribution of anterograde-labeled fibers. PHA-L has been simultaneously injected with the retrograde tracer hydroxystilbamidine (H22845, Polar Tracers—Section 14.3) for double-labeling experiments.
Cholera toxin comprises two subunits, A and B, arranged in an AB5 conformation. Subunit A is an ADP-ribosyltransferase, which disrupts the proper signaling of G proteins and eventually leads to dehydration of the cell. The 12,000-dalton nontoxic subunit B ("choleragenoid"), which is assembled into a 60,000-dalton pentamer above pH 2, allows the protein complex to bind to cellular surfaces via the pentasaccharide chain of ganglioside GM1. All of Molecular Probes cholera toxin subunit B conjugates are prepared from recombinant cholera toxin subunit B, which is completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity.
Cholera toxin subunit B and its conjugates are established as superior tracers for retrograde labeling of neurons. Cholera toxin subunit B conjugates bind to the pentasaccharide chain of ganglioside GM1 on neuronal cell surfaces and are actively taken up and transported; alternatively, they can be injected by iontophoresis. Unlike the carbocyanine-based neuronal tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4), cholera toxin subunit B conjugates can be used on tissue sections that will be fixed and frozen. Our Alexa Fluor 488 (C22841, C34775), Alexa Fluor 555 (C22843, C34776), Alexa Fluor 594 (C22842, C34777) and Alexa Fluor 647 (C34778) conjugates of cholera toxin subunit B combine this versatile tracer with the superior brightness of our Alexa Fluor dyes to provide sensitive and selective receptor labeling and neuronal tracing. We also offer biotin-XX (C34779) and horseradish peroxidase (C34780) conjugates of cholera toxin subunit B for use in combination with diaminobenzidine (DAB) oxidation, tyramide signal amplification (TSA) techniques (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) and Qdot nanocrystal–streptavidin conjugates.
Fluorescent cholera toxins are also markers of lipid rafts—regions of cell membranes high in ganglioside GM1 that are thought to be important in cell signaling. The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405; Tracers for Membrane Labeling—Section 14.4) provide the key reagents, including fluorescent cholera toxin subunit B conjugates, for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes ().
Fluorescent protein–based cytosolic marker BacMam GFP transduction control (B10383) incorporates all the generic advantages of BacMam 2.0 delivery technology (BacMam Gene Delivery and Expression Technology—Note 11.1) to produce nonspecific Green Fluorescent Protein (GFP) expression in a wide range of cell types. Once initiated, GFP expression lasts about 5 days in cell lines such as HeLa and CHO. In cells that divide more slowly or show contact inhibition, such as some stem cells, primary cells and neurons, we have observed useful levels of GFP expression for more than 2 weeks. Within this timeframe, labeled cells can be used for any purpose requiring stable incorporation of a passive fluorescent marker in live cells, including wound healing, migration or adhesion assays. In adoptive transfer applications, cells with undetectable levels of GFP fluorescence can be identified immunohistochemically using our anti-GFP antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4).
Phycobiliproteins make effective polar tracers because they are intensely fluorescent, are very soluble in water and have little tendency to bind nonspecifically to cells (Phycobiliproteins—Section 6.4). These intrinsically fluorescent biomolecules are stable proteins that are monodisperse and substantially larger than albumins, with molecular weights of 240,000 daltons (B- and R-phycoerythrin, P800, P801) and 104,000 daltons (allophycocyanin, A803, or crosslinked allophycocyanin, A819). Phycoerythrin has been conjugated to nuclear-localization peptide sequences to follow nuclear import processes. B-phycoerythrin can pass through pores in erythrocyte membranes created by streptolysin O. Single molecules of R-phycoerythrin have been imaged and tracked within mammalian cells.