Assays for Determination of Protein Concentration

Bradley J.S.C. Olson1

1 Division of Biology, Kansas State University, Manhattan, Kansas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Appendix 3A
DOI:  10.1002/cpph.3
Online Posting Date:  June, 2016
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Abstract

Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high‐throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS‐PAGE sample buffer that is used for equal loading of SDS‐PAGE gels. © 2016 by John Wiley & Sons, Inc.

Keywords: protein assay; SDS‐PAGE; spectrophotometry

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: The Lowry Assay
  • Basic Protocol 2: The Bradford Assay
  • Alternate Protocol 1: Microtiter Plate Bradford
  • Basic Protocol 3: BCA Assay
  • Alternate Protocol 2: Microtiter Plate BCA Assay
  • Basic Protocol 4: UV Absorbance to Measure Protein Concentration
  • Basic Protocol 5: The CBQCA Assay
  • Support Protocol 1: Standard Curves and Data Processing
  • Support Protocol 2: Acetone Precipitation of Protein
  • Support Protocol 3: Trichloroacetic Acid (TCA) Precipitation of Protein
  • Alternate Protocol 3: Alkylation of Reductant and BCA Assay of Protein Samples in SDS‐PAGE Loading Buffer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Lowry Assay

  Materials
  • 1 mg/ml protein standard (e.g., BSA, albumin, or γ‐globulin; see protocol 8)
  • Protein sample
  • Lowry assay mix (see recipe)
  • Freshly prepared diluted Folin‐Ciocalteu reagent (see recipe)
  • Test tubes (e.g., 16 × 125–mm)
  • Spectrophotometer, warmed up and set to 660 nm (or other appropriate wavelength), and cuvette

Basic Protocol 2: The Bradford Assay

  Materials
  • 1 mg/ml protein standard (e.g., BSA, albumin, or γ‐globulin; see protocol 8)
  • Protein sample
  • 1 M NaOH (optional)
  • Bradford reagent (see recipe)
  • Spectrophotometer, warmed up at least 15 min before use and set to 595 nm, and cuvette
  • Test tubes (e.g., 10 × 75–mm for the micro assay or 17 × 100–mm for the macro assay)

Alternate Protocol 1: Microtiter Plate Bradford

  Materials
  • Protein sample
  • 1 mg/ml protein standard (e.g., BSA; see protocol 8)
  • Bradford reagent (see recipe)
  • 1 M NaOH
  • Microtiter plate (see Strategic Planning)
  • Multichannel pipettor or repeating pipettor, optional
  • Microtiter plate reader (see Strategic Planning)

Basic Protocol 3: BCA Assay

  Materials
  • Macro‐BCA or micro‐BCA assay solution (see reciperecipes)
  • Protein samples
  • 1 mg/ml standard proteins (e.g., BSA, albumin, or γ‐globulin; see protocol 8)
  • Test tubes (e.g., 10 × 75–mm for micro‐BCA assay or 13 × 100–mm for macro‐BCA assay)
  • Spectrophotometer, warmed up and set to 562 nm, and cuvette
  • 60°C (or 37°C) heating block or water bath

Alternate Protocol 2: Microtiter Plate BCA Assay

  Materials
  • Micro‐BCA working solution (see recipe)
  • 1 mg/ml protein standard (e.g., BSA; see protocol 8)
  • Microtiter plate(s) (BD Falcon, cat. no. 353915; Thermo Fisher Scientific, Corning Costar 3370, cat. no. 07‐200‐656; Whatman, cat. no. 7701‐1350)
  • Microtiter plate reader, warmed up and ready to read at 562 nm
  • 60°C (or 37°C) oven or water bath

Basic Protocol 4: UV Absorbance to Measure Protein Concentration

  Materials
  • Protein sample
  • Protein standard prepared in the same buffer as unknown
  • Sample buffer
  • Spectrophotometer and quartz or UV‐transparent cuvettes

Basic Protocol 5: The CBQCA Assay

  Materials
  • CBQCA assay kit (Thermo Fisher Scientific, cat. no. C‐6667; kit includes CBQCA reagent, DMSO for dissolving CBQCA reagent, and potassium cyanide; see kit preparation guide below for instructions on preparing the required reagents)
  • 100 mM borate buffer (pH 9.3, see recipe)
  • 100 mM N‐ethylmaleimide (required if assaying protein in the presence of DTT or 2‐mercoptoethanol; see recipe)
  • Protein samples
  • 1 mg/ml standard proteins (e.g., BSA, albumin, or γ‐globulin; see protocol 8)
  • Aluminum foil (to protect the microtiter dish from light)
  • 65°C heating block or oven (optional; used to speed up the reaction)
  • 96‐well microtiter dish (if working with low protein concentrations, dishes should be treated with siliconizing agents to prevent protein absorption to the plastic)
  • Micropipet and tips (low‐binding tips or siliconized tips should be used for dilute protein concentrations)
  • Fluorescence plate reader warmed up and set to excitation wavelength 465 nm and emission wavelength 550 nm; excitation range: 430 to 490 nm, emission range: 550 to 600 nm)

Support Protocol 1: Standard Curves and Data Processing

  Materials
  • 100% acetone, −20°C
  • Protein sample
  • Protein assay‐compatible buffer
  • −20°C freezer
  • 1.5‐ml microcentrifuge tubes (acetone‐compatible, not polycarbonate)
  • Microcentrifuge

Support Protocol 2: Acetone Precipitation of Protein

  Materials
  • 80% (w/v) trichloroacetic acid (TCA)
  • Protein sample
  • 80% acetone, −20°C
  • Protein assay‐compatible buffer
  • −20°C freezer
  • 1.5‐ml centrifuge tubes
  • Microcentrifuge

Support Protocol 3: Trichloroacetic Acid (TCA) Precipitation of Protein

  Additional Materials (also see protocol 4)
  • Stock protein standards
  • 6× SDS‐PAGE loading buffer without bromophenol blue (see recipe)
  • Unknown protein sample
  • 20 mM iodoacetamide solution (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
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   Bensadoun, A. and Weinstein, D. 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70:241‐250. doi: 10.1016/S0003‐2697(76)80064‐4.
   Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal. Biochem. 72:248‐254. doi: 10.1016/0003‐2697(76)90527‐3.
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   You, W.W. , Haugland, R.P. , Ryan, D.K. , and Haugland, R.P. 1997. 3‐(4‐Carboxybenzoyl)quinoline‐2‐carboxaldehyde, a reagent with broad and dynamic range for the assay of proteins and lipoproteins in solution. Anal. Biochem. 244:277‐282. doi: 10.1006/abio.1996.9920.
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