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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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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



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



خصوصیات فیزیکوشیمیایی پروتئینها

  • شکل

  • اندازه

  • بارالکتریکی

Summer School


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


Methods of protein estimation
METHODS OF PROTEIN ESTIMATION involving protein extraction, purification, labeling and analysis.

  • Biuret method

  • Folin- Lowry method

  • Bradford method

  • Bicinchoninic method

  • UV method

  • Flourimetric method

  • Kjeldahl method

  • Mass Spectrometry

Colorimetrc assay

Summer School


Chemistry of protein assays
Chemistry of Protein Assays involving protein extraction, purification, labeling and analysis.

  • 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

Summer School


Biuret test
BIURET TEST involving protein extraction, purification, labeling and analysis.

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

Summer School


Biuret test1
Biuret Test involving protein extraction, purification, labeling and analysis.

  • 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 involving protein extraction, purification, labeling and analysis.

Step 1

Step 2

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Comparison of lowry and biuret
Comparison of Lowry and Biuret involving protein extraction, purification, labeling and analysis.

Summer School


Bicinchoninic method
Bicinchoninic method involving protein extraction, purification, labeling and analysis.

Summer School


Bca test
BCA Test involving protein extraction, purification, labeling and analysis.

  • 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 involving protein extraction, purification, labeling and analysis.

  • 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

Summer School


Dye binding bradford assay1
Dye-Binding ( Bradford ) Assay protein 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.

Summer School



Important criteria for choosing an assay include
Important criteria for choosing an assay include: assay

  • 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

Summer School


Selecting a protein standard
Selecting a Protein Standard assay

  • 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.

Summer School


Examples of standard protein
Examples of Standard Protein assay

  • 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).

Summer School


Standard protein selection
Standard Protein Selection assay

Summer School


Protein to protein variation
Protein-to-Protein Variation assay

  • 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.

Summer School


Methods

Methods assay

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Biosafety in protein assays
Biosafety in protein assays assay

  • Wear Gloves and Labcoat

  • MSDS (Material Safety Data Sheet)

    Folin reagent, Phosphoric acid, ……

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Standard curve

Standard Curve assay

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Standard curve preparation
Standard Curve preparation assay

A750nm

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Comments for standard preparation
Comments for standard preparation assay

  • 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 assay

  • 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

Summer School


Strategies for interfering substance elimination
Strategies for interfering substance elimination assay

  • 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 assay

Summer School


Instrument for bradford assay
Instrument for Bradford assay assay

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Calculations and data analysis note
Calculations and Data Analysis assayNote:

  • 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 assay

  • Direct calculation

    Absorbance values of unknown samples are then interpolated onto the plot

  • Indirect calculation

    formula for the standard curve to determine their concentrations.

Summer School


Indirect calculation
Indirect calculation assay

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Indirect calculation1
Indirect calculation assay

  • C= Concentration

  • OD= Optical Density

  • tgα=Slope of standard curve

  • tgα=∆Cs/∆ODs

  • CX = tgα × ODX

Summer School


Comments
Comments assay

  • 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 assay Specialty to Take Opportunity

Thanks


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