Detection of g protein coupled receptor mediated adenylyl cyclase activity by capillary electrophoresis using fluorescently labeled ATP

A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the detection of G protein coupled receptor mediated adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, cell membranes coexpressing the stimulatory G protein fused to the beta2 adrenergic receptor (beta2AR) and AC were incubated with BATP, the resultant mixture injected, and BATP separated from product BODIPY FL cAMP (BcAMP) by CE. AC activity was quantified by measuring the rate of BcAMP formation. beta2AR agonists isoproterenol and terbutaline increased basal AC activity with EC50s of 2.4 +/- 0.2 and 60 +/- 9 nM, respectively. The antagonist propranolol competed with terbutaline for beta2AR binding sites and expectedly right-shifted the terbutaline dose-response curve to 8 +/- 3 microM. The high sensitivity of the assay was demonstrated by detection of small changes in AC activity, with the partial agonist alprenolol increasing (22 +/- 1%) and the inverse agonist ICI 118,551 decreasing (19 +/- 2%) basal activity. The simplicity and automation of the CE-LIF assay offers advantages over the more traditional assay using radiochemical ATP and column chromatography.     

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF). With FRET, assay signal is the rate of increase in FRET in presence of analyte. With CE-LIF, the assay signal is the peak height of cleavage product formed after a fixed incubation time. The assay has a linear response up to 100 (CE-LIF) or 500 microM (FRET) and detection limit of approximately 500 nM for glucosamine 6-phosphate under single-turnover conditions. When substrate is present in excess of the aptazyme, it is possible to amplify the signal by multiple turnovers to achieve a 13-fold improvement in sensitivity and detection limit of 50 nM. Successful signal amplification requires a temperature cycle to alternately dissociate cleaved substrate and allow fresh substrate to bind aptazyme. The results show that aptazymes have potential utility as analytical reagents for quantification of effector molecules.     

On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies

Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with on-line LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LIF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans G0, G1, G1' and G2.     

Detection of adenylyl cyclase activity using a fluorescent ATP substrate and capillary electrophoresis

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.     

New method based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) to monitor interaction between nanoparticles and the amyloid-β peptide

A novel application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was proposed to efficiently detect and monitor the interaction between polymeric nanoparticles and the β-Amyloid peptide (Aβ(1-42)), a biomarker for Alzheimer's Disease (AD), at concentrations close to physiological conditions. The CE-LIF method allowed the interaction between PEGylated poly(alkyl cyanoacrylate) nanoparticles (NPs) and the soluble Aβ(1-42) peptide monomers to be highlighted. These results were confirmed by surface plasmon resonance (SPR) and confocal laser scanning microscopy (CLSM). Whereas SPR showed an interaction between the NPs and the Aβ(1-42) peptide, CLSM allowed the formation of large aggregates/assemblies at high NP and peptide concentrations to be visualized. All these results suggested that these nanoparticles could bind the Aβ(1-42) peptide and influence its aggregation kinetics. Interestingly, the non-PEGylated poly(alkyl cyanoacrylate) NPs did not alter the aggregation kinetics of the Aβ(1-42) peptide, thus emphasizing the high level of discrimination of the CE-LIF method with respect to NPs.     

Sialic Acid speciation using capillary electrophoresis: optimization of analyte derivatization and separation

Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) was employed for rapid sialic acid speciation, facilitating the quantitative determination of N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) on glycoproteins. Derivatization of the sialic acids with 2-aminoacridone (2-AMAC), using classical reductive amination in a nonaqueous solvent, led to the spontaneous decarboxylation of the sialic acid residues as determined by CE-LIF and offline mass spectrometric analysis. Modification of both the labeling conditions, to drive the decarboxylation reaction to completion and the CE-LIF parameters to separate the neutral species by complexation with a neutral coated capillary and borate reversed polarity, led to a robust platform for the rapid, sensitive, and quantitative speciation of sialic acids. The method can readily be used for quality control of recombinant biopharmaceuticals.     

Electrophoretic profiling of both RNA and protein from a single 250-pL sample

A novel approach is described that uses capillary electrophoresis (CE) to electrophoretically sample and separate both protein and RNA from a single injected plug of cell lysate. A 250-pL sample of lysate from Chinese hamster ovary cells (9.6 x 10(7) cells/mL) was hydrodynamically injected into a capillary containing a Tris-based aqueous buffer. This was followed by selective electrokinetic ejection of RNA from the lysate into water, yielding an effective cell concentration of RNA of 3000 cells/mL. The cellular components (e.g., proteins) retained in the capillary were separated and then detected with laser-induced fluorescence (LIF) using 275-nm excitation. The ejected/diluted sample was subsequently injected into a separate CE-LIF system, which utilized an entangled polymer sieving matrix and 543-nm excitation for the detection of ethidium bromide-labeled nucleic acids (i.e., RNA). Virtually no sample preparation is required other than simple washing and lysing of the cells isolated from culture. This combined approach can be easily modified for the detection of any analyte through adjustment of CE-HF conditions. In addition, it provides an effective method for desalting cellular RNA samples having complex matrixes, which results in improved RNA injection efficiency and a 7600-fold effective signal enhancement over total lysate injection.     

Quantitation of DNA copy number in individual mitochondrial particles by capillary electrophoresis

Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.     

Anomalous radial migration of single DNA molecules in capillary electrophoresis

We report the unexpected radial migration of DNA molecules in capillary electrophoresis (CE) with applied Poiseuille flow. Such movement can contribute to anomalous migration times, peak dispersion, and size and shape selectivity in CE. When Poiseuille flow is applied from the cathode to the anode, DNA molecules move toward the center of the capillary, forming a narrow, highly concentrated zone. Conversely, when the flow is applied from the anode to the cathode, DNA molecules move toward the walls, leaving a DNA-depleted zone around the axis. We showed that the deformation and orientation of DNA molecules under Poiseuille flow was responsible for the radial migration. By analyzing the forces acting on the deformed and oriented DNA molecules, we derived an expression for the radial lift force, which explained our results very well under different conditions with Poiseuille flow only, electrophoresis only, and the combination of Poiseuille flow and electrophoresis. Factors governing the direction and velocity of radial migration were elucidated. Potential applications of this phenomenon include an alternative to sheath flow in flow cytometry, improving precision and reliability of single-molecule detection, reduction of wall adsorption, and size separation with a mechanism akin to field-flow fractionation. On the negative side, nonuniform electroosmotic flow along the capillary or microfluidic channel is common in CE, and radial migration of certain analytes cannot be neglected.     

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.     

Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes whose function in specific aminoacylation of tRNAs is central to the process of protein translation, which occurs in the cytoplasm of all living cells. In addition to their well-established cytoplasmic localization, fluorescence microscopy studies and analysis of the aminoacylation state of nuclear tRNAs have revealed that synthetases are localized in the nuclei of cells from several species including Xenopus laevis and Saccharomyces cerevisiae. Whether nuclear localization of aaRSs is a general phenomenon that occurs in all eukaryotic cells is an open question. In the work described here, human methionyl-tRNA synthetase (MRS) and human lysyl-tRNA synthetase (KRS) were expressed in human-derived DeltaH2-1 osteosarcoma cells as enhanced green fluorescent protein (EGFP) fusion proteins. The subcellular localization of these EGFP-aaRSs was first probed by fluorescence microscopy using cells that coexpressed EGFP-aaRS and a nuclear marker fusion protein, nuDsRed. As expected, both aaRSs were present in the cytosol, while only EGFP-MRS was also clearly localized in the nucleus. To confirm these findings, and to investigate a potentially more sensitive, general method for nuclear localization studies, capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze single DeltaH2-1 cells expressing both EGFP-aaRS and nuDsRed. While cytosolic EGFP signals were detected for both EGFP-MRS and EGFP-KRS, only EGFP-MRS was found in the nucleus, along with nuDsRed. The detection of EGFP-MRS in nuclei of DeltaH2-1 cells demonstrates the feasibility of using CE-LIF analysis in nuclear localization studies of proteins in mammalian cells.     

Quantitative nanopipettor for miniaturized chemical and biochemical reaction sampling

A novel and readily available pipettor capable of nanoliter-sized volume manipulation was developed to improve and increase the flexibility of small-scale reaction processing. The volume delivery was found to be reproducible, with typical relative standard deviations of 1-5%, and easily tunable over a range of nanoliter-sized aliquots. The nanopipettor was combined with capillary electrophoresis using laser-induced fluorescence detection to monitor a small-scale enzyme reaction (beta-galactosidase) using a tetramethylrhodamine-labeled substrate. The results were in good agreement with a standard enzyme assay using a micropipet, thus demonstrating the nanopipettor's potential in developing new nanoscale utrasensitive enzyme assays.     

Distribution of zeptomole-abundant doxorubicin metabolites in subcellular fractions by capillary electrophoresis with laser-induced fluorescence detection

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at < 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.     

Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.     

Determination of Glucose in Submicroliter Samples by CE-LIF Using Precolumn or On-Column Enzymatic Reactions

Two enzymatic reactions combined with capillary electrophoresis (CE) are used to determine glucose contained in sample volumes of ≤500 nL. In the first enzymatic reaction, glucose is oxidized in the presence of glucose oxidase producing hydrogen peroxide, which reacts quantitatively with the fluorogenic compound homovanillic acid catalyzed by the enzyme peroxidase. The second reaction generates a fluorescent species that is proportional to the glucose concentration. The reaction product is determined by CE using laser-induced fluorescence (LIF) as the detection mode. The overall reaction scheme is faster than commonly used precolumn derivatization procedures and can be performed using very small sample quantities. Alternatively, the enzymatic reactions can be performed on-column, similar to the electrophoretically mediated microanalysis approach, accommodating sample quantities in the nanoliter range. The on-column reaction is a simple and practical approach for the determination of glucose contained in low-volume samples by CE-LIF, where samples are injected directly into the capillary column without any pretreatment. However, sample handling and detectability of the precolumn approach proved to be superior. Determination of glucose using the precolumn and on-column reactions showed detection limits of 50 and 800 nM, respectively. The methods were shown to be linear in the range tested, 1-100 μM and 100 nM-30 μM, for the on-column and precolumn reactions, respectively. The reproducibility for each scheme was <5% RSD. To determine the possibility of using a noninvasive procedure for glucose monitoring, we used the CE-LIF methods to analyze human tear samples for glucose. The tear fluid samples were contained in a volume of ～200 nL. The concentration of glucose in the human tear samples analyzed using the precolumn and on-column procedures was ～138 μM.     

Detection of green fluorescent protein in a single bacterium by capillary electrophoresis with laser-induced fluorescence

Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis.     

Coaxial flow system for chemical cytometry

Over the past decade, chemical cytometry performed by capillary electrophoresis (CE) has become increasingly valuable as a bioanalytical tool to quantify analytes from single cells. However, extensive use of CE-based chemical cytometry has been hindered by the relatively low throughput for the analysis of single adherent cells. In order to overcome the low throughput of CE-based analysis of adherent cells and increase its utility in evaluating cellular attributes, new higher throughput methods are needed. Integration of a coaxial buffer exchange system with CE-based chemical cytometry increased the rate of serial analyses of cells. In the designed system, fluid flow through a tube coaxial to the separation capillary was used to supply electrophoretic buffer to the capillary. This sheath or coaxial fluid was turned off between analysis of cells and on during cell sampling and electrophoresis. Thus, living cells were not exposed to the nonphysiologic electrophoretic buffer prior to lysis. Key parameters of the system such as the relative capillary-sheath positions, buffer flow velocities, and the cell chamber design were optimized. To demonstrate the utility of the system, rat basophilic leukemic cells loaded with Oregon green and fluorescein were serially lysed and loaded into a capillary. Separation of the contents of 20 cells at a rate of 0.5 cells/min was demonstrated.     

Accurate DNA fragment sizing by capillary electrophoresis with laser-induced fluorescence array for detection of sequence specificity of DNA damage

Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a [...GGCGCGCAG...] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene. This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with (32)P end labeling.     

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.     

Affinity assays using fluorescence anisotropy with capillary electrophoresis separation

A novel approach to detecting affinity interactions that combines fluorescence anisotropy with capillary electrophoresis (FACE) was developed. In the method, sample is injected into a capillary filled with buffer that contains a fluorescent probe that possesses low fluorescence anisotropy. If proteins or other large molecules in the sample bind the fluorescent probe, their migration through the capillary can be detected as a positive anisotropy shift. Thus, the method provides both separation and confirmation of binding to the probe. Calculations based on combining the Perrin equation and dissociation constant were used to predict the effect of conditions on aniostropy detection. These calculations predict that low probe concentrations yield the best sensitivity while higher concentrations increase the dynamic range for detection of binding partner. The assay was applied to detection of G proteins using BODIPY FL GTPgammaS as the fluorescent probe. Experimental measurements exhibited trends in anisotropy with varying probe and protein concentrations that were consistent with the calculations. The limit of detection for G(alphai1) was 3 nM when the electrophoresis buffer contained 250 nM BODIPY FL GTPgammaS. FACE affinity assay is envisioned as a method that can quantify selected binding partners and screen complex samples for compounds that possess affinity for a particular small molecule that is used as a probe.