Protein AssayTechnical HandbookTable of Contents Total Protein AssaysQuick Technical Summaries ......................................3Introduction..........................................................6Selection of the Protein Assay......................................................................6Selection of a Protein Standard....................................................................7Standards for Total Protein Assay ................................................................8Sample Preparation ....................................................................................9Protein:protein Variation ............................................................................9Compatible and Incompatible Substances ................................................10Compatible Substances Table ..................................................................11Time Considerations ................................................................................13Calculation of Results ..............................................................................13BCA™-based Protein Assays ....................................14Chemistry of the BCA™ Protein Assay ......................................................14Advantages of the BCA™ Protein Assay ....................................................15Disadvantages of the BCA™ Protein Assay................................................16BCA™ Protein Assay..................................................................................17Micro BCA™ Protein Assay ......................................................................18BCA™, Reducing Agent Compatible ..........................................see page 24Coomassie Dye-based Protein Assays (Bradford Assays) 19Chemistry of Coomassie-based Protein Assays ........................................19Advantages of Coomassie-based Protein Assays ......................................19Disadvantages of Coomassie-based Protein Assays ................................20General Characteristics of Coomassie-based Protein Assays ....................20Coomassie Plus – The Better Bradford™ Assay ........................................21Coomassie (Bradford) Protein Assay ........................................................22Coomassie Dry Protein Assay Plates ........................................................22Overcoming Interfering Substances............................23BCA™, Reducing Agent Compatible ..........................................................24Compat-Able™ Protein Assays ..................................................................25Modified Lowry Protein Assay ..................................26Chemistry of the Modified Lowry Protein Assay ........................................26Advantages of the Modified Lowry Protein Assay ......................................27Disadvantages of the Modified Lowry Protein Assay..................................27General Characteristics of the Modified Lowry Protein Assay....................27Modified Lowry Protein Assay ..................................................................28Amine Detection ..................................................29OPA Fluorescent Protein Assay..................................................................29Fluoraldehyde o-Phthaladehyde ................................................................30Specific Protein AssaysHistidine-Tagged Proteins ......................................31HisProbe™ HRP ........................................................................................31SuperSignal®West Pico HisProbe™ Kit ....................................................31Antibodies ..........................................................32Easy-Titer®IgG and IgM Assays................................................................32Proteases ..........................................................34QuantiCleave™ Protease Assays................................................................34Glycoproteins ......................................................35Glycoprotein Carbohydrate Estimation Assay ............................................35Phosphoproteins ..................................................36Phosphoprotein Phosphate Estimation Assay ............................................36PhosphoProbe™-HRP................................................................................37Peroxide ............................................................38PeroXOquant™ Quantitative Peroxide Assay..............................................384For more product information, or to download a product instruction booklet, visit www.piercenet.com/path95n.Quick Technical Summaries – Total Protein AssaysCoomassie Plus – The Better Bradford™ AssayWorking RangeLinear Range:IgG: 125-1,500 g/mlBSA: 125-1,000 g/mlStandard Assay:Sample-to-ReagentRatio: 1:30Typical Working Range: 100-1,500 g/mlMicro Assay:Sample-to-ReagentRatio: 1:1Typical Working Range: 1-25 g/mlCoomassie (Bradford) Protein AssayWorking RangeStandard Assay:Sample-to-ReagentRatio: 1:50100-1,500 g/mlMicro Assay:Sample-to-ReagentRatio: 1:11-25 g/mlCharacteristics/AdvantagesSimple/fast protocolsTotal preparation and assay time <30 minutesOne reagent system; stable for 12 monthsReady-to-use formulation — no dilutionor filtration neededNearly immediate color development at room temperature Linear color response in standard assay (more accurate results)Color response sensitive to changes in pHTemperature dependence of color responseCompatible with buffer salts, metal ions, reducing agents, chelating agentsLow-odor formulationCharacteristics/AdvantagesSimple-to-perform protocolsOne-reagent system, stable for 12 monthsReady-to-use formulationNo dilution or filtration neededFast, nearly immediate color development at room temperatureTotal preparation and assay time <30 minutesTypical protein:protein variation expected for a Coomassie dye-based reagentColor response sensitive to pHTemperature-dependent color responseCompatible with buffer salts, metal ions, reducing agents, chelating agentsApplicationsStandard assay8Micro assay9,10,11Microplate format assay12Assay of protein solutions containing reducing agents13Quantitation of immobilized protein14Protein in permeabilized cells15NaCNBH3 determination16ApplicationsStandard assay8Micro assay9,10,11Microplate format assay19Assay of protein solutions containing