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Graphical abstract, Luciferase-based reporter assays are one of the most common cell-based screening formats for drug discovery, and simultaneous evaluation of the cytotoxic effect of test compounds is of great value in reducing false-positives. Here we share a multiplex assay protocol that allows sequential measurement of cell viability (cell number) and luciferase activity of the same sample in a multi-well-plate format. The viability assay employs a fluorogenic esterase substrate, CytoRed. • This protocol allows sequential measurement of endogenous esterase activity (as a surrogate for cell number) and then luciferase activity in a single sample. • The protocol eliminates the need for parallel viability assay or protein assay using separate aliquots of the lysate. • This protocol is especially useful for assays with cells stably expressing a luciferase construct, for which co-transfection of another reporter gene is not a viable option.

SUMMARY Introduction The reduced susceptibility of biofilms to disinfectants presents a challenge to the successful reprocessing of medical equipment. This study examined the effect of residual biomass remaining after previous disinfection with peracetic acid (PAA) on the tolerance of subsequent mature Pseudomonas aeruginosa biofilms to PAA. The effect of enzymatic degradation of specific components of the extracellular polymeric substance (EPS) of P. aeruginosa biofilm on the effectiveness of PAA disinfection was also evaluated. Methods The susceptibility of biofilm grown on the biomass of PAA-killed biofilm to PAA was compared with the PAA susceptibility of biofilm grown in wells of a 24-well plate by evaluating their viability using the plate count assay. The effect of PAA on biofilm biomass was measured using crystal violet quantification of total biofilm biomass, while its effect on the polysaccharide and protein components of biofilm EPS was quantified using the phenol-sulphuric acid assay or Bradford assay, respectively. A confocal microscope was used to visualize the distribution of living and dead cells in biofilms grown on residual biofilm biomass. Findings The presence of residual biomass from previously disinfected biofilms significantly enhanced the tolerance of subsequent biofilms. A 96-h-old ‘secondary biofilm’ formed on disinfected biomass survived PAA concentrations of 4000 ppm, which exceeds the concentrations used in practice for high-level disinfection. Conclusion These observations indicate that, under certain circumstances, recolonization of residual EPS can cause failure of disinfection of medical equipment such as endoscopes, and emphasizes the importance of cleaning endoscopes prior to disinfection.

Background Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. Aim Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. Method We performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. Result Precision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3–102.4%, 0.3–3.8 ​mg/mL, and 1.2–12.8 ​mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 ​± ​16.2 ​mg/mL and 2.1 ​± ​1.5 ​mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 ​> ​0.6). Conclusion Paper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples., Highlights • Thorough evaluation of colorimetric protein assays on paper-analytical device. • Bromocresol green and tetrabromophenol blue methods were found to be best suited for serum and urine samples, respectively. • The selected methods are comparable with spectrophotometric assay protocols for human serum and urine samples.

The effects of antibacterial compounds produced by Saccharomyces cerevisiae in Koumiss on pathogenic Escherichia coli 0 8 and its cell surface characteristics were investigated. S. cerevisiae isolated from Koumiss produced antibacterial compounds which were active against pathogenic E. coli O 8 as determined by the Oxford cup method. The aqueous phases from S. cerevisiae at pH=2.0 (S2) and pH=8.0 (S8) were extracted and tested, respectively. The organic acids of S2 and S8 were determined by high performance liquid chromatography (HPLC), and the concentrations of killer toxins were determined by enhanced bicinchoninic acid (BCA) Protein Assay Kit. The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of S2 and S8 on E. coli O 8 were determined by the broth microdilution method. The effects of S2 and S8 on the growth curve of E. coli O 8 were determined by turbidimetry, and the hydrophobicities of E. coli O 8 cell surface were determined using the microbial adhesion to solvents method, the permeation of E. coli O 8 cell membrane were determined by the o-nitrophenyl-β-D-galactoside (ONPG) method. Aqueous phases at pH 2.0 and 8.0 had larger inhibition zones and then S2 and S8 were obtained by freeze-drying. The main component in S2 was citric acid and it was propanoic acid in S8. Other organic acids and killer toxins were also present. Both the MICs of S2 and S8 on E. coli O 8 were 0.025 g mL −1 , the MBCs were 0.100 and 0.200 g mL −1 , respectively. The normal growth curve of E. coli O 8 was S-shaped, however, it changed after addition of S2 and S8. E. coli O 8 was the basic character, and had a relatively hydrophilic surface. The hydrophobicity of E. coli O 8 cell surface and the permeation of E. coli O 8 cell membrane were increased after adding S2 and S8. The present study showed that S2 and S8 inhibit the growth of pathogenic E. coli O 8 and influence its cell surface characteristics.

Small molecule ligands, which can bind to a protein of interest with high affinity and specificity, are a burgeoning kind of recognition element in nano-inspired biosensors for protein assay. Similar to most other recognition molecules mentioned in previous Chapters, small molecule ligands are also unable to respond to target protein binding in a measurable way. To resolve this problem, rationally designed and engineered small molecule ligand-based probes, such as small molecule-tethered chemical probes and small molecule-linked DNA probes, have been rapidly developed and found successful applications in detecting disease protein biomarkers. This chapter aims to provide an overview of the latest advances in this field by detailing a number of representative biosensors according to the category of small molecule ligand-based probes.

In this work, we present an enzyme-free electrochemical assay method that can detect target protein with ultra-high sensitivity through dual amplification. Firstly, target protein is specifically captured by its aptamer, and then is released due to toehold-mediated click chemical ligation (the first round of recycling) via DNA strand displacement reaction. Secondly, the overhang of aptamer on the electrode surface can hybridize with RP DNA and trigger hybridization chain reaction (the second round of recycling). Consequently, large amount of electrochemical species hexaammineruthenium (III) chloride ([Ru(NH3)6]3+) can be embedded into double-stranded DNA to produce a remarkable electrochemical signal, thus the target protein can be quantified with ultra-high sensitivity. Taken thrombin as a model analyte, a wide linear dynamic range from 100 fM to 10 nM and a detection limit of 30 fM (S/N = 3) can be obtained. Meanwhile, since no enzyme is required for the measurement, the assay is relatively simple and inexpensive. Therefore, the protein assay method proposed in this work may have a great potential for clinical diagnosis and biomedical research in the future. Keywords: Protein assay, Electroanalysis, Enzyme-free, Click chemical ligation, Hybridization chain reaction

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used.

To minimize oxidative modification, a low tem- perature, sequential flotation method was developed to isolate plasma lipoproteins in 18 h using a benchtop ultra- centrifuge. The protein distributions were characterized using agarose and SDS-polyacrylamide gel electrophore- sis, and an SDS-Lowr y protein assay. The lipid distribu- tions were assessed using a gas chromatography-mass spec- trometric assay for cholesterol and an enzymatic assay for triglycerides. To validate the rapid flotation method, lipoproteins were also isolated from the same plasma samples using a modified Havel et al. flotation method ( J. Clin. Invest. 34: 1345-1353, 1955). The same lipoproteins and apolipoproteins were present in fractions of compa- rable density, and the summed recoveries of protein, cho- lesterol, and triglyceride were also identical for the Havel et al. and rapid fl otation procedures. Likewise, the amount of cholesterol and triglyceride in corresponding very low, intermediate, and low density lipopr otein (VLDL/IDL and LDL) fractions was the same for the two flotation procedures. The triglyceride and cholesterol lev- els in high density lipoprotein (HDL) isolated by rapid flo- tations, however, were 9-12% higher than in the HDL as isolated by Havel et al. Because a 9 -12% increase in the HDL fraction reflects only 1- 4% of the total triglyceride and cholesterol in plasma, we conclude that, while main- tained at 4 8 C, lipoproteins were quantitatively isolated from human plasma in 1 day.— Tong, H., H. R. Knapp, and M. VanRollins. A low temperature flotation method to rapidly isolate lipoproteins from plasma. J. Lipid. Res. 1998. 39: 1696-1704.

Publisher Summary Single-cell protein (SCP) refers to protein derived from cells of microorganisms such as yeast, fungi, algae, and bacteria, which are grown on various carbon sources for synthesis. This chapter discusses the present status of microbial SCP production from agricultural wastes and describes some of the technical and economical problems related to the production processes that must be overcome for large-scale application. The dried cells of microorganisms or the whole organism is harvested and consumed. SCP is a protein source for human food supplements and animal feeds. SCP production may have potential for feeding the ever-increasing world population. Massive quantities of SCP can be produced in a single day. As a source of protein it is very promising with potential to satisfy the world shortage of food while population increases. There are several carbon sources that are used as energy sources for microorganisms for growing and producing SCP.

Publisher Summary This article describes techniques for the preparation and partial purification of three different loaded endosomal fractions from BHK cells: early endosomes, endosomal carrier vesicles, and late endosomes. Dissolve 20 mg of biotin-XNHS in 0.5 ml dimethylformamide. Mix by adding the biotin dropwise to the HRP mixture while gently stirring or shaking the Erlenmeyer and incubate at room temperature with gentle stirring for at least 45 min. Intact microtubules are required for the delivery of endocytosed markers to the LE. Therefore, markers accumulate in transport intermediates (ECVs) in the absence of microtubules. Whereas stable microtubules are cold sensitive, dynamic microtubules are depolymerized easily in the presence of nocodazole. It is important that cells are homogenized under conditions where endosomes are released from cells, yet where latency is high so that the endosomes are not broken and retain their internalized marker. Measure the amount of protein in each gradient fraction using a standard protein assay system, as described in the manual.

Publisher Summary This chapter describes the in vitro assay that was developed for the faithful formation, isolation, and characterization of COPI-derived vesicles from highly purified rat liver Golgi membranes. Although the exact mechanism for vesicle formation and cargo incorporation is still debated, the fundamental coat components, as well as the basic principles of vesicle formation, have been clear for some time. The hydrolysis of GTP by ADP-ribosylation factor 1 (Arfl), stimulated by a GTPase-activating protein (GAP), is then thought to trigger vesicle uncoating, producing a transport intermediate capable of fusing with target membranes. After terminating the reaction, slowly sedimenting vesicles are separated from heavier donor membranes by medium speed centrifugation. The COPI-derived vesicles are then recovered using high speed centrifugation onto a two step sucrose cushion. Protein concentration determination is performed using the Bio-Rad version of the Bradford protein assay reagent and BSA as a standard. Pipetting of Golgi membranes should be minimized to avoid shearing. Mix these aliquots by tapping carefully on the side of the tube.

This chapter describes the purification of several Rabkinesin-6 domains from bacteria and eukaryotic systems, and their biochemical characterization (ATPase, MT-binding, hydrodynamic properties).The chapter describes an assay to visualize the interaction between Rab6A and Rabkinesin-6. For purification, Escherichia coli strain freshly transformed with pET-Nt or pTrc-Q665 plasmids are cultured. Bacterial pellets are obtained are sonicated, centrifuged, and chromatographed. Protein fractions are analyzed by SDS-PAGE and Western blot. This protocol leads to the purification of about 0.5–1 mg fusion proteins. α/β-Tubulin is purified from bovine brain with two cycles of polymerization in the presence of GTP, and microtubule (MT)-associated proteins (MAPs) are removed by phosphocellulose chromatography. Tubulin concentration is estimated by the Bradford assay and MT concentration is expressed per tubulin heterodimer. MT-induced ATPase activity of kinesins is often measured by using a NADH coupled enzymatic assay.

A variety of methods like sonication, homogenization with a glass or Teflon pestle, grinding with aluminum or glass beads, and high pressurization and rapid release of pressure are used for cell-extract preparation as the extraction of cellular material from whole cells is a critical step in enzyme purification. The preparation of cell extracts (also called cell-free extracts) requires a very careful execution so as to ensure complete release of the enzyme from cellular material without denaturation of the enzyme itself. In case of yeast, the easiest and most effective method for cell-extract preparation involves grinding with glass beads. The shearing action produced by the glass beads during vortexing causes the rupture of the yeast cell. The glass beads grinding procedure is currently used as the most effective technique for yeast cell extraction as the very thick and tough cell wall of yeast renders methods—such as sonication, osmotic lysis, and homogenization—ineffective in breaking the cell. The Bradford protein assay technique has been further elaborated in this chapter.

Publisher Summary This chapter focuses on three techniques that can be used to determine the protein content of tissues or samples. The three techniques include Lowry Assay, Bradford Assay, and Neuhoff Assay. All assays described in this article quantitate protein relative to a standard protein. The choice of the standard protein can markedly influence the result. This requires special attention for proteins with a high content of certain amino acids. The most efficient way to prepare an exact dilution series of the standard protein employs a handheld dispenser. Many reagents used commonly in protein extraction interfere with Lowry assay. The dot-blot assay is a versatile tool. Its different modifications enable one to cope with almost every potentially interfering substance. The dot-blot assay combines high sensitivity, an extended range of linearity, and high tolerance to potentially interfering substances. The Lowry assay exhibits the best accuracy with regard to absolute protein concentrations due to the chemical reaction with polypeptides. It is also useful for the quantitation of oligopeptides.

Publisher Summary This chapter describes the procedures for preparation and characterization of one type of versatile and stable liposomes as well as for covalent conjugation of neoglycoproteins and lectins to the liposomes, and characterization of the conjugates. To analyze the homogeneity and stability of liposomes, size distribution is an important characteristic among several physical properties. Gel-permeation chromatography (GPC), electron microscopy (EM), and dynamic light scattering (DLS) can be applied for size characterization of liposomes. The chapter discusses the procedure for GPC analysis. A GPC result is compared with analyses by EM and DLS. To analyze the purity and stability of neoglycoprotein-coupled liposomes or lectin-coupled liposomes, GPC analysis is routinely carried out by using the high-performance liquid chromatography (HPLC) system. To analyze the yield and quality of neoglycoprotein-coupled liposomes or lectin-coupled liposomes, protein and lipid compositions in each preparation are routinely determined, and coupled protein/total lipid ratios calculated. The protein content of neoglycoprotein-liposome and lectin-liposome conjugates is estimated by either employing the modified Lowry method in the presence of 1% sodium dodecyl sulfate, or by using a commercial micro-bovine serum albumin (BSA) protein assay kit in the presence of 1% sodium dodecyl sulfate.

Publisher Summary This chapter describes the physicochemical characteristics of the 5-HT3 receptor and the different methods used to purify the 5-HT3 receptor. The successful solubilization of 5-HT3 receptor-binding sites can be achieved from an abundant source using a large spectrum of detergents and salt conditions. However, physicochemical studies require more restrictive conditions. The successful solubilization of membrane-bound receptor proteins requires an abundant source of receptors. The chapter reviews the properties of the most common biological detergents and highlights their respective advantages and disadvantages. The solubilization of the 5-HT3 receptor-binding sites allows the determination of several of their physical properties and leads to the purification of the ligand-binding protein(s). Proteins are estimated using the bicinchoninic acid (BCA) protein assay reagent with BSA as a standard. The physicochemical properties of amino acid sequence of the 5-HT3 receptor-binding subunit are altogether consistent with 5-HT3 receptor being a member of the superfamily of ionophoric receptors.

Publisher Summary This chapter highlights ultrasmall gold probes. It describes its characteristics and uses in immuno (cyto) chemical studies. Markers are used either directly bound to the antigen-specific antibody (direct method) or linked to bridging molecules that bind specifically to the primary antibody (indirect method). Colloidal gold is preferred to radioactive or enzyme markers because of its particulate nature, which results in improved resolution. In addition, gold probes are nonhazardous reagents. Colloidal gold particles carry a net negative surface charge because of ions that are adsorbed on the surface of the particles. Positively charged proteins can therefore bind electrostatically. This phenomenon has been exploited to stain protein bands on blots and to develop a sensitive protein assay. Various considerations are taken into account when trying to improve the performance of colloidal gold probes, such as sensitivity of an immunogold reaction and sensitivity of detection. Gold probes based on ultrasmall particles are called “ultrasmall gold probes.” Contrary to the larger sized particles, uncoated ultrasmall colloidal gold particles are not stable and will aggregate in time. However, after adsorption to proteins the particles are stabilized and maintain their original size.

Publisher Summary This chapter describes a procedure using Tar-containing membranes and purified soluble chemotaxis proteins that demonstrates a role for the Tar chemoreceptor and the CheW protein in modulating the phosphorylation reactions. Additionally, reference to heterologous systems using chimeras made by fusing portions of Tar to other receptors proteins for testing their effect on phosphate transfer reactions and/or signaling are presented for comparison. For protein assay, the total protein concentration of each preparation is measured using the Bio-Rad protein reagent concentrate, with bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is performed by using 12.5% slab resolving gels. The position of proteins on the gel is determined by Coomassie staining or Western blot analysis. CheW is expressed from the plasmid pDV4 in strain KO685. For purification of CheW, strain KO685 with the pDV4 plasmid is cultivated in LB containing 100 μg/ml ampicillin at 37 ° until logarithmic phase. Tar-containing membranes are isolated from a strain containing a plasmid which overexpresses the protein using a tac promotor P. Plasmids expressing mutant Tar proteins are constructed by replacement of a restriction fragment containing the wild-type sequence with a corresponding mutant fragment. In this way, both "tumble" and "smooth" receptor-encoding vectors are constructed. These mutant Tar proteins proved useful in verification of the specificity of the activation of phosphorylation. All plasmids are maintained in a strain deleted of all four Escherichia coli chemoreceptors. This strain is also used as a source of negative control membranes.

Publisher Summary This chapter discusses a technology used for conducting comparative exocellular forms of the enzyme extracellular superoxide dismutase (EC-SOD) therapy of the physiological diseases of the mutants. Human EC-SOD is a tetrameric glycoprotein, whose molecular weight is 135,000. The chapter describes purification procedures for selecting proteins of this nature. In SOD assay, the spectrophotometric method of Misra and Fridovich is used. The method is based on the inhibition by the enzyme of the spontaneous, alkali-catalyzed autoxidation of epinephrine to norepinephrine. In protein assay, the spectrophotometric method of Murphy and Kies is used. The course of dialysis to remove tetraborate is monitored by a colorimetric assay. A sample of the dialysate (0.9 ml) is mixed with 0.1 ml of 1% silver nitrate and incubated at room temperature for about 10 minutes or until the absorbancy at 500 nm becomes constant. The extinction coefficient of tetraborate is 0.255. The assay detects as little as 10–100 μ g sodium tetraborate per milliliter. The purification procedures are based on the assumption that all EC-SODs are large, highly glycosylated proteins. The literature on highly glycosylated proteins indicates that their properties resemble that of polysaccharides and sugars. Solubility and resistance to denaturation in organic solvents is one feature of highly glycosylated proteins exhibited by EC-SODs. They are soluble and remain active in 80–95% ethanol. The yeast enzyme is also soluble and fully active after extraction in 80% phenol. Furthermore, unlike ordinary protein, EC-SODs are soluble in aqueous saturated ammonium sulfate.

Publisher Summary This chapter discusses the assays for superoxide dismutase (SOD) based on the autoxidation of hematoxylin. The assay is based on the transformation of hematoxylin to its two-electron-oxidized product, hematein. The assay is sensitive and simple. The reaction product, hemarein, is relatively stable and has an absorption maximum and extinction coefficient similar to those of the commonly used O 2 – scavenger, cytochrome c . The reaction is inhibited by SOD and can be performed in the physiological pH range of 6.8–7.8, where SOD inhibits the reaction 90–95%. In addition, at pH values above 8.1, the autoxidation reaction is accelerated by added SOD, and the reaction becomes a positive assay for the enzyme, similar in principle to SOD assays based on the photochemical and enzymatic oxidation of dianisidine. The activity of purified SOD is determined by the xanthine oxidase cytochrome c method. Xanthine oxidase is isolated from unpasteurized cream. General protein is determined by the Bradford assay using bovine serum albumin (BSA) as a standard.

Publisher Summary This chapter describes a method that is capable of accurately measuring low amounts of a protein in the presence of very high levels of lipid. This procedure was developed from the amido black 10 B methods of Schaffner and Weissmann and Newman et al. and incorporates several critical modifications that enable an assay to be performed with lipid-containing samples without any interference. One approach has been to remove an interfering lipid by extraction with organic solvents. However, because certain proteins display a limited solubility in such solvents, this strategy often fails. Another widely used approach involves the inclusion of sodium dodecyl sulfate (SDS) in a modified Lowry procedure to reduce lipid (and detergent) interference. As oxidized lipid continues to react to produce a substantial amount of color in the Lowry assay and as most lipid samples are partially oxidized, this procedure is not suitable for the accurate measurements of a protein in samples containing excess of lipid.

Publisher Summary The Lowry and Bradford assays for protein determination provide relatively simple methods for calculating specific activities by standardizing enzyme activities to protein content. Both assays are colorimetric. The Lowry assay depends on the complexing of copper with tyrosine and tryptophan residues, whereas the Bradford method measures the binding of Coomassie Brilliant Blue G-250 to protein. The protocols are modified specifically for protein determination in Triton lysates but may be altered for different conditions by simply replacing Triton with the appropriate agent. The standards may be plotted on a linear graph of concentration versus absorbance, and unknown concentrations may then be read directly from the graph. Alternatively, a straight line may be fit to the standard data by the method of least squares and the formula generated may be used to calculate unknown concentrations. This latter method is especially convenient for use with a hand-held programmable calculator. The blank cuvette should be rinsed approximately every 10 min to avoid progressive staining of the cuvette.

Publisher Summary This chapter discusses the measurement of protein in lipid extracts. In the course of studies of organic-solvent-soluble proteins, a variety of classic protein assay methods have been found to be inapplicable or involving excessive effort to reduce interference as a result of the presence of lipids in the mixture. The simplest resource, extracting the lipids away from the protein by the use of organic solvents, fails because of the solubility of the protein in organic solvents. The chapter explores that 2,4,6-trinitrobenzene sulfonic acid (TNBS) of a suitable quality is obtained commercially as a pale yellow crystalline material. It may be recrystallized from 1 M HCl or from 2-butanone-petroleum ether mixtures. As the crystalline material may have a variable degree of hydration, the stock solution of 0.03 M TNBS in butanol (tertiary : secondary, 9:1 v/v) is conveniently diluted from a stronger solution after an alkalimetric check on its concentration. Organic solvents and potassium tetraborate are the analytical reagent grade or equivalent. The 6 M HCl used is the easily prepared 6.1 M azeotrope; a dilution of 12 M HCl would be equally satisfactory.

Publisher Summary This chapter describes the procedure for the purification of the proton-translocating ATPase from rat liver mitochondria using the detergent 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). The important feature of the procedure that follows for the purification of the F 0 F 1 -ATPase from rat liver mitochondria that distinguishes it from other procedures of purification of F 0 F 1 ATPase complexes is the use of the detergent CHAPS that is developed as an alternative to existing detergents. CHAPS appears to be the detergent of choice for the solubilization of the membrane-bound F 0 F 1 ATPase complex. A change in aggregation of the complex forms the basis for the purification of the F 0 F 1 ATPase complex. This modification simplifies the isolation procedure and eliminates the problems that occur when trying to control for the presence of sucrose in the aliquots assayed for protein concentration via the Bradford protein assay. The purified and reconstituted complex catalyzes ATP-P i exchange and ATP-dependent proton translocation.

Publisher Summary This chapter describes the biotin-binding protein present in egg yolk. The assay is based on the temperature-accelerated exchange of bound biotin for free [ 14 C]biotin. The procedure is, therefore, useful for biotin-binding proteins with no free binding sites. The purification procedure for biotin-binding protein of egg yolk is described in the chapter. All buffers used are potassium phosphate, pH 7.2, at the concentration given. The procedure is for 5 dozen eggs. The eggs are cracked and the white and yolks are separated. The yolks are broken and 1.5 times the yolk volume in distilled water is added. The diluted yolk is well stirred. This solution is centrifuged at 13,200 g for 30 minutes. The supernatant is collected, and solid NaCl is added to make the solution 1 M in salt. After the salt has completely dissolved, the solution is poured into centrifuge bottles, two-third the solution volume in 1-butanol is added, and the mixture is shaken vigorously. The centrifugation of the resulting emulsion at 13,200 g for 15 minutes causes a lipid-free aqueous phase and a lipid-rich organic phase to form, with a solid layer of precipitated protein at the interface. The chapter discusses the properties of the biotin-binding protein.