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Protein Quantification Methods

- May 05, 2017

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Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins (e.g., for detecting proteins, for isolating and purifying proteins, and for characterizing the structure and function of proteins, often requiring that the protein first be purified). Computational methods typically use computer programs to analyze proteins. However, many experimental methods (e.g., mass spectrometry) require computational analysis of the raw data.


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Genetic methods

Experimental analysis of proteins typically requires expression and purification of proteins. Expression is achieved by manipulating DNA that encodes the protein(s) of interest. Hence, protein analysis usually requires DNA methods, especially cloning. Some examples of genetic methods include conceptual translation, Site-directed mutagenesis, using a fusion protein, and matching allele with disease states. Some proteins have never been directly sequenced, however by translating codons from known mRNA sequences into amino acids by a method known as conceptual translation. (See genetic code.) Site-directed mutagenesis selectively introduces mutations that change the structure of a protein. The function of parts of proteins can be better understood by studying the change in phenotype as a result of this change. Fusion proteins are made by inserting protein tags, such as the His-tag, to produce a modified protein that is easier to track. An example of this would be GFP-Snf2H which consists of a protein bound to a green fluorescent protein to form a hybrid protein. By analyzing DNA alleles can be identified as being associated with disease states, such as in calculation of LOD scores.


Protein Quantification Methods Video



Protein extraction from tissues

Protein extraction from tissues with tough extracellular matrices (e.g., biopsy samples, venous tissues, cartilage, skin) is often achieved in a laboratory setting by impact pulverization in liquid nitrogen. Samples are frozen in liquid nitrogen and subsequently subjected to impact or mechanical grinding. As water in the samples becomes very brittle at these temperature, the samples are often reduced to a collection of fine fragments, which can then be dissolved for protein extraction. Stainless steel devices known as tissue pulverizers are sometimes used for this purpose.

Advantages of these devices include high levels of protein extraction from small, valuable samples, disadvantages include low-level cross-over contamination.


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Protein purification

  • Protein isolation
    • Chromatography methods: ion exchange, size-exclusion chromatography (or gel filtration), affinity chromatography
  • Protein extraction and solubilization
  • Concentrating protein solutions
  • Gel electrophoresis
    • Gel electrophoresis under denaturing conditions
    • Gel electrophoresis under non-denaturing conditions
    • 2D gel electrophoresis
  • Electrofocusing

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Detecting proteins

Non-specific methods that detect total protein only

  • Absorbance: Read at 280 or 205 nm. Can be very inaccurate. Detection in the range of 100 ?g/mL to 1 mg/mL. Ratio of absorbance readings taken at 260/280 can indicate purity/contamination of the sample (pure samples have a ratio <0.8)
  • Bradford protein assay: Detection in the range of ~1 mg/mL
  • Biuret Test Derived Assays:
    • Bicinchoninic acid assay (BCA assay): Detection down to 0.5 ?g/mL
    • Lowry Protein assay: Detection in the range of 0.01-1.0 mg/mL
  • Fluorescamine: Quantifies proteins and peptides in solution if primary amine are present in the amino acids
  • Amido black: Detection in the range of 1-12 ?g/mL
  • Colloidal gold: Detection in the range of 20 - 640 ng/mL
  • Nitrogen detection:
    • Kjeldahl method: used primarily for food and requires oxidation of material
    • Dumas method: used primarily for food and requires combustion of material

Specific methods which can detect amount of a single protein

  • Spectrometry methods:
    • High-performance liquid chromatography (HPLC): Chromatography method to detect proteins or peptides
    • Liquid chromatography-mass spectrometry (LC/MS): Can detect proteins at low concentrations (ng/mL to pg/mL) in blood and body fluids, such as for Pharmacokinetics.
  • Antibody dependent methods:
    • Enzyme-linked immunosorbent assay (ELISA): Specifically can detect protein down to pg/mL.
    • Protein immunoprecipitation: technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
    • Immunoelectrophoresis: separation and characterization of proteins based on electrophoresis and reaction with antibodies.
    • Western blot: couples gel electrophoresis and incubation with antibodies to detect specific proteins in a sample of tissue homogenate or extract (a type of Immunoelectrophoresis technique).
    • Protein immunostaining

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Protein structures

  • X-ray crystallography
  • Protein NMR
  • Cryo-electron microscopy
  • Small-angle X-ray scattering

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Interactions involving proteins

  • Protein footprinting

Protein-protein interactions

  • (Yeast) two-hybrid system
  • Protein-fragment complementation assay
  • Co-immunoprecipitation
  • Affinity purification and mass spectrometry

Protein-DNA interactions

  • ChIP-on-chip
  • Chip-sequencing
  • DamID
  • Microscale thermophoresis

Protein-RNA interactions

  • Toeprinting assay
  • TCP-seq

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Computational methods

  • Molecular dynamics
  • Protein structure prediction
  • Protein sequence alignment (sequence comparison, including BLAST)
  • Protein structural alignment
  • Protein ontology (see gene ontology)



Other methods

  • Hydrogen-deuterium exchange
  • Mass spectrometry
  • Protein sequencing
  • Protein synthesis
  • Proteomics
  • Peptide mass fingerprinting
  • Ligand binding assay
  • Eastern blotting
  • Metabolic labeling
    • Heavy isotope labeling
    • Radioactive isotope labeling

Source of the article : Wikipedia



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