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Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

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Thermo Scientific Pierce Protein Assay Technical Handbook Version 2 Table of Contents Total Protein Assays Specialty Assays Quick Technical Summaries 1 Histidine-tagged Proteins 32 Thermo Scientific HisProbe-HRP Kits 32 Introduction 4 Selection of the Protein Assay 4 Antibodies 33 Selection of a Protein Standard 5 IgG and IgM Assays 33 Standard Preparation 6 Proteases 35 Standards for Total Protein Assay 7 Compatible and Incompatible Substances 9 Protease Assays 35 Compatible Substances Table 10 Glycoproteins 36 Time Considerations 12 Glycoprotein Carbohydrate Estimation Assay 36 Calculation of Results 12 Phosphoproteins 37 Thermo Scientific Pierce 660nm Protein Assay 13 Phosphoprotein Phosphate Estimation Assay 37 Overview 13 Highlights 13 Peroxides 38 Typical Response Curves 14 Quantitative Peroxide Assay 38 BCA-based Protein Assays 15 Spectrophotometers 39 Chemistry of the BCA Protein Assay 15 BioMate 3S UV-Visible Spectrophotometer 39 Advantages of the BCA Protein Assay 16 Evolution 260 Bio UV-Visible Spectrophotometer 39 Disadvantages of the BCA Protein Assay 17 Evolution 300 UV-Visible Spectrophotometer 40 BCA Protein Assay – Reducing Agent Compatible 18 Evolution Array UV-Visible Spectrophotometer 40 BCA Protein Assay 19 Micro BCA Protein Assay 20 Coomassie Dye-based Protein Assays (Bradford Assays) 21 Chemistry of Coomassie-based Protein Assays 21 Advantages of Coomassie-based Protein Assays 21 Disadvantages of Coomassie-based Protein Assays 21 General Characteristics of Coomassie-based Protein Assays 22 Coomassie Plus (Bradford) Protein Assay 23 Coomassie (Bradford) Protein Assay 24 Removal of Interfering Substances 25 Thermo Scientific Compat-Able Protein Assays 26 Modified Lowry Protein Assay 27 Chemistry of the Modified Lowry Protein Assay 27 Advantages of the Modified Lowry Protein Assay 28 Disadvantages of the Modified Lowry Protein Assay 28 General Characteristics of the Modified Lowry Protein Assay 28 Modified Lowry Protein Assay Reagent 29 Amine Detection 30 OPA Fluorescent Protein Assay 30 Fluoraldehyde o-Phthaladehyde 31 Quick Technical Summaries – Thermo Scientific Protein Assays Working Range (sample volume)* Characteristics/Advantages Applications Disadvantages Interfering Substances Pierce® 660nm Protein Assay Standard Protocol: Compatible with reducing agents, chelating Ideal for measuring total Use reagent with IDCR High levels of ionic 25-2,000µg/mL (65µL) agents and detergents protein concentration in (Ionic Detergent detergents require the samples containing Compatibility Reagent) addition of the Ionic Microplate Protocol: Faster and easier to perform than BCA or both reducing agents with samples containing Detergent Compatibility 50-2,000µg/mL (10µL) Coomassie (Bradford) Assays and detergents ionic detergents like SDS Reagent (IDCR). Excellent linearity of color development within Used for quick, yet accurate Greater protein-to-protein the detection range estimation of protein variability than the Less protein-to-protein variability than the BCA Assay Coomassie (Bradford) Assay Reaches a stable end point Compatible with Laemmli sample buffer containing bromophenol blue when using Compatibility Buffer The BCA Protein Assay - Reducing Agent Compatible Standard Protocol: Compatible with up to 5mM DTT, 35mM Allows the use of the Requires heating for Compatible with all reducing 125-2,000µg/mL (25µL) 2-Mercaptoethanol or 10mM TCEP superior BCA Assay in color development agents and detergents found situations in which it is at concentrations routinely Microplate Protocol: No protein precipitation involved normally unable to be read used in protein sample buffers 125-2,000µg/mL (9µL) Sample volume only 9µL (microplate protocol) No precipitation step Compatible with most detergents means no worries about difficult-to-solubilize proteins Significantly less (14-23%) protein:protein variation than Bradford-based methods The BCA Protein Assay Standard Protocol: Two stable reagents used to make one Adaptable for use with Not compatible with Reducing sugars and 20-2,000µg/mL (50µL) working reagent microplates thiols/reducing agents reducing agents Enhanced Standard Working reagent stable for one week at Determine the amount of Requires heating for Thiols Protocol: 5-250µg/mL room temperature IgG coated on plates color development Copper chelating agents (50µL) Compatible with detergents Measure the amount of Not a true end-point assay Ascorbic acid and uric acid Microplate Protocol: protein covalently bound Simple, easy to perform 20-2,000µg/mL (25µL) to affinity supports Tyrosine, cysteine and Less protein:protein variation than Coomassie tryptophan Determine copper levels dye methods using a reagent formulated 50mM Imidazole, 0.1M Tris, Works with peptides (three amino acids with BCA Reagent A4 1.0M glycine or larger) Flexible incubation protocols allow customization of reagent sensitivity and working range The Micro BCA Protein Assay Standard Protocol: Three stable reagents used to make one Suitable for determining More substances interfere Reducing sugars and 60°C for 60 minutes working reagent protein concentration at lower concentrations reducing agents 0.5-20µg/mL (0.5mL) in very dilute aqueous than with BCA Assay Working reagent stable for 24 hours at Thiols solutions because the sample Microplate Protocol: room temperature volume-to-reagent volume Copper chelating agents 37°C for 120 minutes Adaptable for use with Compatible with most detergents ration is 1:1 2-40µg/mL (150µL) microplates1 Ascorbic acid and uric acid Simple, easy to perform 60°C water bath is needed Tyrosine, cysteine and Less protein:protein variation than BCA, tryptophan Coomassie dye or Lowry Methods 50mM Imidazole, 0.1M Tris, Works with peptides (three amino acids 1.0M glycine or larger) Linear color response to increasing protein concentration * Sample volume per 1mL total assay volume for measurement in 1cm cuvette (Standard Protocol). Sample volume per 200-300µL total volume for measurement in 96-well microplate. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1 Quick Technical Summaries – Thermo Scientific Protein Assays Working Range (sample volume)* Characteristics/Advantages Applications Disadvantages Interfering Substances The Modified Lowry Protein Assay Standard Protocol: Two-reagent system – shelf life of at least Lowry method is the most Timed addition of Folin Detergents 1-1,500µg/mL one year cited protein assay in the reagent adds complexity (cause precipitation) literature Microplate Protocol: Two-step incubation requires precise Longer total assay time Thiols, disulfides 10-1,500µg/mL (40µL) sequential timing of samples Adaptable for use with Practical limit of about Copper chelating reagents microplates Color response read at 750nm 20 samples per run Carbohydrates including Works with peptides (three amino acids hexoseamines and their or larger) N-actyl derivatives Protein:protein variation similar to that seen Glycerol, Tris, Tricine, K1+ ions with BCA Method Many authors have reported ways to deal with substances that interfere Coomassie Plus (Bradford) Assay Linear Range: Simple/fast protocols Standard assay8 Less linear color response Detergents18 IgG: 125-1,500µg/mL in the micro assay Total preparation and assay time < 30 minutes Micro assay9,10,11 BSA; 125-1,000µg/mL Effect of interfering One reagent system; stable for 12 months Microplate format assay12 Standard Protocol: substances more Sample-to-Reagent Ready-to-use formulation — no dilution or Assay of protein solutions pronounced in the Ratio: 1:30 filtration needed containing reducing agents13 micro assay Typical Working Range: Nearly immediate color development at Quantitation of immobilized Protein dye complex has 100-1,500µg/mL (35µL) room temperature protein14 tendency to adhere to Microplate Protocol: Linear color response in standard assay glass (easily removed Protein in permeabilized Sample-to-Reagent (more accurate results) with MeOH)17 cells15 Ratio: 1:1 Color response sensitive to changes in pH 16 Protein must be > 3,000 Da Typical Working Range: NaCNBH3 determination 1-25µg/mL (150µL) Temperature dependence of color response Compatible with buffer salts, metal ions, reducing agents, chelating agents Low-odor formulation Coomassie (Bradford) Protein Assay Standard Protocol: Simple-to-perform protocols Standard assay8 Nonlinear color response Detergents18 Sample-to-Reagent One-reagent system, stable for 12 months Micro assay9,10,11 More protein standard Ratio: 1:50 concentrations required to 100-1,500µg/mL (20µL) Ready-to-use formulation Microplate format assay19 cover working range Microplate Protocol: No dilution or filtration needed Assay of protein solutions Micro assay has potential Sample-to-Reagent containing reducing agents Fast, nearly immediate color development at for interference Ratio: 1:1 room temperature Cell line lysates20 1-25µg/mL (150µL) Protein must be > 3,000 Da Total preparation and assay time < 30 minutes Protein recovery studies Typical protein:protein variation expected for a Coomassie dye-based reagent Color response sensitive to pH Temperature-dependent color response Compatible with buffer salts, metal ions, reducing agents, chelating agents * Sample volume per 1mL total assay volume for measurement in 1cm cuvette (Standard Protocol). Sample volume per 200-300µL total volume for measurement in 96-well microplate. 2 For more information, or to download product instructions, visit www.thermoscientific.com/pierce Quick Technical Summaries
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