Health & Research

Methods of Protein Characterization

Need to characterize Protein:

Protein characterization is necessary in biological development and in research:

  • To determine protein structure
  • To determine the actual amino acid composition
  • To sequence of the protein product
  • To examine the post-translation modifications on the proteins which can affect their functions
  • To determine disulfide bonds

Steps for protein characterization:

  • Cloning
  • Expression
  • Purification
  • Characterization

Expression cloning:

  • A technique in DNA cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein.

An example would be using an expression library to isolate genes that could confer antibiotic resistance.

  Expression vector:

  • A specialized type of cloning vector in which the transcriptional and translational signals needed for the regulation of the gene of interest are included in the cloning vector.
  • The transcriptional and translational signals may be synthetically created to make the expression of the gene of interest easier to regulate.

Purpose of Expression cloning:

The aim of expression cloning is

  • to produce large quantities of specific proteins
  • to enhance translation of the gene of interest’s mRNA
  • a transcription termination sequence,
  • in eukaryotes specific sequences to promote the post-translational modifications of the protein product.

Protein purification:

  • A series of processes to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.
  • Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.
  • The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins.
  • The pure result may be termed protein isolate.

Protein Separation & Detection

  • Separate proteins with 1D or 2D Gel Electrophoresis
    • Isoelectric focusing
      • Immobilized pH gradients (IPG DryStrip)
      • Tube gel (carrier Ampholytes)
    • SDS PAGE
      • Reduced and alkylated Stain proteins
  • Protein Characterization:

3D Structure Determination:

Can be done either by

X-ray crystallography or

NMR ( nuclear magnetic resonance)

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