In the name of god
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In the name of God. Summer School. Influenza Unit, Pasteur Institute of Iran summer 2012. PROTEINS Assay Methods (Protein quantitation ). B.Farahmand. Summer School. INTRODUCTION.

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In the name of god

In the name of God


Summer school

Summer School

Influenza Unit,

Pasteur Institute of Iran

summer 2012


Proteins assay methods protein quantitation

PROTEINSAssay Methods(Protein quantitation)

B.Farahmand

Summer School


Introduction

INTRODUCTION

  • Proteins are highly complex natural compounds composed of large number of different amino acids.


Amino acids

Amino acids

Summer School


In the name of god

Summer School


Levels of protein organization

Levels of Protein Organization

  • Primary structure = linear chain of amino acids

  • • Secondary structure = domains of repeating structures, such as β-pleated

  • sheets and α-helices

  • • Tertiary structure = 3-dimensional shape of a folded polypeptide, maintained by disulfide bonds, electrostatic interactions, hydrophobic effects

  • • Quaternary structure = several polypeptide chains associated together to form a functional protein

Summer School


In the name of god

Summer School


In the name of god

خصوصیات فیزیکوشیمیایی پروتئینها

  • شکل

  • اندازه

  • بارالکتریکی

Summer School


In the name of god

Protein Estimation is a part of any laboratory workflow involving protein extraction, purification, labeling and analysis.


Methods of protein estimation

METHODS OF PROTEIN ESTIMATION

  • Biuret method

  • Folin- Lowry method

  • Bradford method

  • Bicinchoninic method

  • UV method

  • Flourimetric method

  • Kjeldahl method

  • Mass Spectrometry

Colorimetrc assay

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Chemistry of protein assays

Chemistry of Protein Assays

  • Copper-based Protein Assays:

    • Biuret Protein Assays

    • Lowry Assay

    • BCA

      Protein-copper chelation and secondary detection of the reduced copper

  • Dye-based Protein Assays:

    • Coomassie (Bradford) Assay

      Protein-dye binding and direct detection of the color change associated with the bound dye

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Biuret test

BIURET TEST

By reducing the copper ion from cupric to cuprous form, the reaction produces a faint blue-violet color

Summer School


Biuret test1

Biuret Test

  • Adventage

  • Reproduciple

  • Very few interfering agents

    (ammonium salts being one such agent )

  • Fewer deviations than with the Lowry or ultraviolet absorption methods

  • Disadventage

  • Requires large amounts protein (1-20mg)

  • Low sensitivity

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Folin ciocalteu lowry assay

Folin-Ciocalteu ( Lowry ) Assay

Step 1

Step 2

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Comparison of lowry and biuret

Comparison of Lowry and Biuret

Summer School


Bicinchoninic method

Bicinchoninic method

Summer School


Bca test

BCA Test

  • Adventage

  • The color complex is stable

  • There is less suceptibility to detergents

  • Fewer deviations than with the Lowry or Beradford methods

  • Disadventage

  • Bicinchonic acid is expensive

Summer School


Dye binding bradford assay

Dye-Binding ( Bradford ) Assay

  • CBBG primarily responds to arginine residues

  • (eight times as much as the other listed residues)

  • If you have an arginine rich protein,

  • You may need to find a standard

  • that is arginine rich as well.

  • CBBG binds to these residues in the anionic form

  • Absorbance maximum at 595 nm (blue)

  • The free dye in solution is in the cationic form,

  • Absorbance maximum at 470 nm (red).

  • Bradford, MM. A rapid and sensitive for the quantitation of microgram

  • quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.

  • Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

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Mechanism of dye response and interference in the bradford protein assay

Mechanism of Dye response and interference in the Bradford protein assay

Anionic dye

Protonated or cationic amino acids

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Dye binding bradford assay1

Dye-Binding ( Bradford ) Assay

  • Adventage

  • Fast and inexpensive

  • Highly specific for protein

  • Very sensitive [1-20 µg (micro assay) 20-200 µg (macro assay)]

  • Compatible with a wide range of substances

  • Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin)

  • Dye reagent complex is stable for approximately one hour

  • Disadventage

  • Non-linear standard curve over wide ranges

  • Response to different proteins can vary widely, choice of standard is very important

Summer School


Comparison of standard curve of bradford lowry and bca assay

Comparison of standard curve of Bradford, Lowry and BCA assay

  • Absorption spectra of anionic and cationic forms of the dye overlap.

    So the standard curve is non-linear.

  • The assay performs linearly over short concentration stretches.

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Selecting a protein assay a standard protein

Selecting a Protein Assay & a Standard protein

Summer School


Important criteria for choosing an assay include

Important criteria for choosing an assay include:

  • Compatibility with the sample type and components

  • Assay range and required sample volume

  • Protein-to-protein variation

  • Speed and convenience for the number of samples to be tested

  • Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay

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Selecting a protein standard

Selecting a Protein Standard

  • If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve in the selected protein assay method and is readily available to any laboratory at any time.

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Examples of standard protein

Examples of Standard Protein

  • Generally, bovine serum albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively inexpensive.

  • Alternatively, bovine gamma globulin (BGG) is a good standard when determining the concentration of antibodies because BGG produces a color response curve that is very similar to that of immunoglobulin G (IgG).

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Standard protein selection

Standard Protein Selection

Summer School


Protein to protein variation

Protein-to-Protein Variation

  • Each protein in a sample responds uniquely in a given protein assay. Such protein-to-protein variation refers to differences in the amount of color (absorbance) obtained when the same mass of various proteins is assayed concurrently by the same method.

    These differences in color response relate to differences in:

    - amino acid sequence,

    - isoelectric point (pI),

    - secondary structure

    - and the presence of certain side chains or prosthetic groups.

  • Depending on the sample type and purpose for performing an assay, protein-to-protein variation is an important consideration in selecting a protein assay method and in selecting an appropriate assay standard (e.g., BSA vs. BGG). Protein assay methods based on similar chemistry have similar protein-to-protein variation.

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Methods

Methods

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Biosafety in protein assays

Biosafety in protein assays

  • Wear Gloves and Labcoat

  • MSDS (Material Safety Data Sheet)

    Folin reagent, Phosphoric acid, ……

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Standard curve

Standard Curve

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Standard curve preparation

Standard Curve preparation

A750nm

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Comments for standard preparation

Comments for standard preparation

  • For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed.

  • This is particularly true for the protein assay methods that produce non-linear standard curves.

  • Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required.

  • In general, fewer points are needed to construct a standard curve if the color response is linear.

  • Typically, standard curves are constructed using at least two replicates for each point on the curve.

Summer School


Sample preparation for protein assays

Sample Preparation for Protein Assays

  • it must be solubilized

  • inhibit microbial growth

  • avoid casual contamination of the sample by foreign debris such as dust, hair, skin or body oils.

  • After filtration or centrifugation to remove the cellular debris, typical samples will still include nucleic acids, lipids and other non-protein compounds.

  • nonprotein components (detergents, biocides or antimicrobial agents , protease inhibitors, different salts, denaturants, reducing agents and chaotropes) are critical for choosing an appropriate assay

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Strategies for interfering substance elimination

Strategies for interfering substance elimination

  • Choose a different protein assay method or a version of the same assay method that includes components to overcome the interference.

  • Dialyze or desalt the sample to remove interfering substances that are small (i.e., less than 1000 daltons), such as reducing agents.

  • Precipitate the protein in TCA or other appropriate reagent, remove the solution containing the interfering component, and then redissolve the protein for analysis.

Summer School


Instrument for lowery assay

Instrument for Lowery assay

Summer School


Instrument for bradford assay

Instrument for Bradford assay

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Calculations and data analysis note

Calculations and Data AnalysisNote:

  • With most protein assays, sample protein concentrations are determined by comparing their assay responses to that of a dilution-series of standards whose concentrations are known. Protein samples and standards are processed in the same manner by mixing them with assay reagent and using a spectrophotometer to measure the absorbances. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations.

Summer School


Unknown sample concentration calculation

Unknown sample concentration calculation

  • Direct calculation

    Absorbance values of unknown samples are then interpolated onto the plot

  • Indirect calculation

    formula for the standard curve to determine their concentrations.

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Indirect calculation

Indirect calculation

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Indirect calculation1

Indirect calculation

  • C= Concentration

  • OD= Optical Density

  • tgα=Slope of standard curve

  • tgα=∆Cs/∆ODs

  • CX = tgα × ODX

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Comments

Comments

  • Obviously, the most accurate results are possible only when unknown and standard samples are treated identically. This includes assaying them at the same time and in the same buffer conditions, if possible. Because different pipetting steps are involved, replicates are necessary if one wishes to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error.

  • Although most modern spectrophotometers and plate readers have built-in software programs for protein assay data analysis, several factors are frequently misunderstood by technicians. Taking a few minutes to study and correctly apply the principles involved in these calculations can greatly enhance one's ability to design assays that yield the most accurate results possible (see the related Tech Tips and links).

Summer School


Aquire specialty to take opportunity

Aquire Specialty to Take Opportunity

Thanks


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