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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

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

slide9
خصوصیات فیزیکوشیمیایی پروتئینها
  • شکل
  • اندازه
  • بارالکتریکی

Summer School

slide10

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

Summer School

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

Summer School

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

Summer School

folin ciocalteu lowry assay
Folin-Ciocalteu ( Lowry ) Assay

Step 1

Step 2

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).

Summer School

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
  • 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:
  • 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
  • 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
  • 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

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.

Summer School

methods

Methods

Summer School

biosafety in protein assays
Biosafety in protein assays
  • Wear Gloves and Labcoat
  • MSDS (Material Safety Data Sheet)

Folin reagent, Phosphoric acid, ……

Summer School

standard curve

Standard Curve

Summer School

standard curve preparation
Standard Curve preparation

A750nm

Summer School

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

Summer School

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

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.

Summer School

indirect calculation1
Indirect calculation
  • C= Concentration
  • OD= Optical Density
  • tgα=Slope of standard curve
  • tgα=∆Cs/∆ODs
  • CX = tgα × ODX

Summer School

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

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