|Courtesy of BD PharMingen|
Multicolor immunofluorescent cell staining showing cell surface FITC (green) staining and intracellular PE (orange) and APC (red) staining.
Fluorescence technology tries at times to be all things to all people, and the researcher usually must make a complex choice about protein detection. To help with the first--and difficult--step of selecting an available assay or deciding to design one, the general avenues of fluorescence-based protein detection technologies will be explored. At the very minimum, this should fill the gaps in most researchers' intellectual databases of fluorescence information and may save countless research hours duplicating assays or creating reagents available with a single phone call.
Fluorescent probes (fluorophores) receive energy from photons produced by an incandescent lamp or a laser, exciting some of their electrons. This transient configuration loses some energy within electronic orbitals but goes back to the ground energy state through the emission of a photon (fluorescence). Collisional quenching can dissipate fluorescence by increasing the nonradiative component. The photon released by fluorescence has less energy than the exciting one; thus excitation spectra occur at shorter wavelengths than emission spectra. This wavelength difference between excitation and emission is called Stokes' shift. The greater the separation between these wavelengths, and thus the Stokes' shift, the lower the background. Multiple parameters can be examined in a single experiment by using probes with different emission spectra such as coumarin (blue fluorescence), fluorescein (green), and rhodamine (orange-red).
All Things Immunofluorescent
For any protein, fluorescence-based assays can be developed based on specific recognition of the protein surface by an antibody; immunofluorescence technology allows visualization and detection both on surfaces (blots, tissue sections) or the cellular milieu. The ideal reagent is a fluorophore-conjugated antibody that binds specifically to the protein being characterized (primary antibody). Roche Molecular Biochemicals offers M30 Cyto DEATH, a fluorescein-labeled antibody that binds to a caspase-related cleavage product of cytokeratin 18; this antibody recognizes apoptotic cells. Chemicon International has developed several double-labeling approaches for virologists. SimulFluor™ detects herpes simplex virus type 1 (HSV-1)-infected versus HSV-2-infected cells by dual labeling; another version differentiates HSV and varicella zoster virus (VZV) infection. This company also has a line of products known as Light Diagnostics for cytomegalovirus or Pneumocystis carinii detection.
|Courtesy of Zymed Laboratories|
Indirect immunofluorescence detection of claudin-3 in rat small intestine using a FITC-conjugated secondary antibody from Zymed Labs.
Immunofluorescence is usually performed indirectly through secondary detection. A primary antibody (unlabeled) binds to the protein antigen, and the complex is visualized with a fluorophore-conjugated secondary antibody. This avoids chemical labeling of precious monoclonal antibodies or concerns about a decrease in immunoreactivity of labeled primary antisera. Secondary antibodies can be labeled with dozens of different fluorophores, elected based on the detection process chosen and the number of antigens to be detected (double or multiple labeling). Many manufacturers offer fluorophore-conjugated secondary antibodies that recognize specific antigen-antibody complexes. Zymed Laboratories' antiimmunoglobulin antibodies, for example, are available tagged with FITC, Cy™3 and Cy5 cyanine dyes (em~565 and 667 nm, respectively), Texas Red®, and tetramethylrhodamine conjugates. CALBIOCHEM provides conjugates to FITC, rhodamine, PE, and Texas Red®. Roche Molecular Biochemicals adds fluorescein- or PE-labeled F(ab')2 affinity- purified fragments. Molecular Probes has a broad array of fluorophore-modified secondary antibodies, with the classic fluorescein, rhodamine, eosin, R-PE, B(blue)-PE, and also novel dyes such as Alexa™ (em~603 or 617 nm), which gives brighter fluorescence than either rhodamine or Cy3.
Biotinylated proteins or antibodies react specifically with avidins: egg white avidin or streptavidin from Streptomyces avidinii. These proteins have four biotin-binding subunits and are used instead of a fluorescent secondary antibody. A number of companies offer fluorophore-conjugated avidin and streptavidin. Some of the fluorescent dyes are proprietary, such as Life Technologies' RED670®, a fluorophore consisting of a cyanine derivative combined with R-PE, and RED613®, a tandem probe of Texas Red® and R-PE. These RED probes avoid autofluorescence problems and allow three-color analysis with R-PE and fluorescein. A large selection of fluorescently labeled avidin and streptavidin is available from Molecular Probes, featuring a broad range of fluorescence emissions and two dozen fluorophores.
Biotin-based immunodetection can also target cell-bound antigens using specific ligands. Flow cytometry provides the means to detect and sort the cells carrying labeled ligand-bound protein. R&D Systems' Fluorokine™ system employs biotin-conjugated cytokines to label cell surface receptors, and avidin-FITC reveals receptor-cytokine complexes. This design can be adapted to almost any cellular system in which a specific protein ligand is known, using available kits for biotinylation of proteins (American Qualex and Sigma offer such tools), and the large strept/avidin family of fluorescent conjugates described.
Enzymatic Activity Revealed
Fluorescence methods are also being used quite elegantly for enzymatic studies. Enzymes can be exposed to substrates that change their intrinsic fluorescence when processed by the target protein. Typically, the substrate is nonfluorescent and converted to a fluorophore through one or more reactions. Molecular Probes offers kits based on the Amplex™ red reagent, a stable and sensitive nonfluorescent probe that is oxidized by hydrogen peroxide (H2O2) to highly fluorescent resorufin (em~587 nm). These kits combine peroxidating secondary reactions and can detect glucose, monoamine and glutamate oxidases, acetylcholinesterases, PC-specific phospholipase C, phospholipase D, and sphingomyelinase. CLONTECH Laboratories offers Great EscAPE™, a fluorescent assay for secreted alkaline phosphatase (SEAP); SEAP is a popular reporter for monitoring the effect of gene promoter elements. Great EscAPE uses an AP-sensitive fluorogenic substrate, 4-methylumbelliferul phosphate (MUP). The assay is linear over a 10,000-fold range of enzyme concentration and exhibits a sensitivity comparable to luciferase-based assays.
The substrate can also contain fluorophores but remain nonfluorescent until attacked by the target enzyme; this is accomplished by substrate design, incorporating several neighboring self-quenching fluorophores. A standard protocol is to label a protein with many copies of a fluorophore. Proteolytic activity is measured by release of small fluorescent peptides. Molecular Probes has developed an entire family of fluorescence protease assays based on this principle (EnzCheck™). The most general of these assays uses casein heavily labeled with pH-insensitive BODIPY® FL or BODIPY TR-X (green and red fluorescence, respectively). This assay detects metallo, serine, acid, and sulfhydryl proteases; it avoids cumbersome trichloroacetic acid precipitation steps and can be optimized for high-throughput screening (HTS). Another EnzCheck assay uses fluorescein-labeled DQ™ gelatin to measure extracellular matrix metallo proteases (MMPs, including gelatinases, collagenases, and stromelysins) that digest collagen, gelatin, and other components of the extracellular matrix. MMPs are released when cells invade a neighboring tissue, both in health (wound healing) and disease (metastasis). An elastase EnzCheck, optimizable for HTS, uses DQ elastin labeled with BODIPY FL.
Another way to study cleavage of a substrate is to combine a quencher and a fluorophore on the same molecule. Rupture of intramolecular bonds separates the fluorophore and quencher and leads to emission of fluorescence. Research Diagnostics Inc. uses this principle in its CONFLUOLIP™ assay, a test for analysis of triglyceride hydrolysis in postheparin plasma and tissue fluids. These kits (available for lipoprotein and hepatic lipases) use a triglyceride with an added pyrene proximal to a quenching trinitrophenyl group. In the presence of active lipase, the quencher is hydrolyzed and the pyrene becomes fluorescent (em~400 nm).
This general scheme can also be applied to enzymes that cleave oligonucleotides. FRETWorks™ from Novagen uses an RNase substrate that becomes highly fluorescent upon enzyme cleavage. The substrate has a fluorophore at the 5'-end and a quencher at the 3'-end. As the fluorophore is released, it becomes highly fluorescent. FRETWorks enables very sensitive detection of RNase A with potential for HTS applications.
The activity of protein kinases can also be studied by fluorescence methods. Protein kinases were classically assayed by measuring the transfer of radioactive phosphate to a substrate protein or peptide; the large amount of 32P needed made this assay inconvenient and potentially hazardous. Promega offers a nonisotopic solution with its PepTag® assay for protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The PepTag Assay uses fluorescent peptide substrates that are highly specific for each kinase. Phosphorylation by PKC or PKA alters the peptide net charge from +1 to -1, allowing separation of phosphorylated and nonphosphorylated versions of the substrate on agarose gels at neutral pH. Less than 10 ng of kinase can be detected in under two hours. Remarkably, phosphorylation of substrates other than the peptides does not alter the results (a nagging problem with radioactive detection), and very low backgrounds are obtained even with impure samples.
Fluorescent detection of NEN's Fluo-somatostatin bound to the sst2A somatostatin receptor (upper) and amplified with TSA-Direct Cyanine 3 (lower).
An antigen-antibody complex is typically formed with a 1:1 stoichiometry, making antigen detection with fluorescent antibodies less sensitive than with enzyme-linked antibodies that produce luminescent or chromogenic (colored) products. Many contemporary assays in immunohistochemistry or immunodetection (e.g., ELISA assays) use luminescent reactions or chromogenic systems employing horseradish peroxidase (HRP) or alkaline phosphatase (AP). However, enzymatic reactions can be coupled with fluorogenic substrates to amplify the signal originating from a single antigen-antibody complex. These types of reactions are called chemifluorescent and can be considered "conversion technologies," as they allow conversion of traditional HRP-, AP-, or chemiluminescence-based assays to fluorescence. The high sensitivity and linearity over a wide dynamic range exhibited by chemifluorescence assays make them an attractive alternative.
Several chemifluorescence assays have simply coupled HRP and AP with fluorogenic substrates. An AP fluorescent system offered by Promega, AttoPhos®, uses a prefluorescent substrate that is cleaved by AP into inorganic phosphate and a highly fluorescent benzothiazole anion. This fluorescence enhancement is maximized by this probe's unusually large Stokes' shift (120 nm), leading to very low backgrounds and subpicomolar (attomole) detection levels. AttoPhos converts AP-based ELISA assays to fluorescence. For conversion of HRP-based assays, two options are available. QuantaBlu™ from Pierce Chemical Company effectively replaces colorimetric HRP substrates with a blue fluorogenic substrate (em~420 nm). This substrate has almost ideal properties: It does not photobleach, allows determinations of HRP down to 1 pg, produces a linear fluorescent signal over four linear log decades, and possesses a Stokes' shift of 95 nm, leading to excellent signal-to-noise ratios. HRP can be detected very rapidly (0.62 pg in 6.5 minutes) with ensuing applications in stopped-flow kinetics. Another HRP fluorescence assay system is the Amplex system from Molecular Probes, using H2O2-reactive probes to detect HRP activity. This assay exhibits low autofluorescence interference and can be used for HRP-based fluorescence ELISAs.
Another realm in which fluorescence technologies are replacing chromogenic substrates is surface detection (immunohistochemistry and in situ hybridization). NEN Life Science Products has created the Renaissance® TSA technique, enhancing detection 100-fold. TSA technology uses HRP to catalyze the deposition of biotin- or fluorescently labeled tyramide onto tissue sections or cell preparations previously blocked with proteins. Added labels are deposited proximal to the enzyme site, resulting in minimal loss of resolution and significant enhancement of the fluorescent signals. TSA kits use direct staining with tyramide (conjugated with coumarin, fluorescein, tetramethylrhodamine, or Cyanine 3 or 5) or indirect staining with biotinyl-tyramide. The biotinyl-tyramide can be detected fluorescently with antifluorescein HRP or antifluorescein AP plus streptavidin conjugated with fluorescein or indirectly with a chromogenic substrate. The availability of four fluorophore-conjugated tyramides enables sequential, multianalyte detection protocols or combination of protein and nucleic acid detection on the same slide.
Back to Basics
The previous sections consider fluorescent detection by virtue of protein shape (epitopes) or activity. But fluorescent methods are also being used to replace standard protein quantitation methods. Molecular Probes provides two such basic systems suitable for HTS applications. NanoOrange® protein quantitation kit measures 20 ng to 2 µg of protein in a microplate format, an order of magnitude more sensitive than standard colorimetric assays. The reagent changes from nonfluorescent to fluorescent in the presence of detergent-coated proteins (em~590 nm). The CBQCA kit provides a fluorogenic reagent that reacts with amines in proteins; it is particularly useful for quantification of lipid-protein mixtures or lipoproteins, replacing unstable fluorescamine-based methods. The reagent is nonfluorescent until exposed to cyanide and protein, and the resulting fluorescence emission at 570-590 nm is in the detection range of many fluorometers or microplate readers.
Researchers can now even engineer proteins to contain endogenous fluorogenic signals. In the FRETWorks™ S*Tag™ Assay Kit from Novagen, a protein of interest is fused to a 15-amino acid S*Tag peptide. The fusion protein activates S protein to form active RNase S. The active enzyme is provided with an RNase-specific substrate with a fluorophore on the 5'-end and a quencher on the 3'-end. Upon cleavage of the molecule, the fluorophore is released and becomes brightly fluorescent. This method allows specific and extremely sensitive detection of S*Tag fusion proteins in minutes. Another S*Tag Assay technology for detection of S*Tag fusion proteins uses S protein-FITC conjugates to reveal the protein; it provides an ideal way to stain insect or mammalian cells to confirm recombinant protein expression.
Despite the multitude of fluorescence detection methods and applications described, it is safe to say that the power of this approach has not been fully exploited. Stay tuned for advances in miniaturization, multiparameter analysis, single-molecule detection, and robotics.
Jorge D. Cortese can be contacted at firstname.lastname@example.org.