Answer to Question #250364 in Biochemistry for Bonolo

Question #250364
Describe the principles behind five (5) laboratory techniques used in the isolation and
analysis of proteins
Expert's answer
  1. Preparation of crude extracts: Effective removal of the entire protein from the preliminary material is essential for the achievement of any purification process. The process is done for the whole disruption of cells and issue of contents from cellular wreckage.
  2. Precipitation of proteins: Available methods for precipitation of proteins utilize changes in pH and temperature, or adding of salts and animate solvents. For example, ammonium sulfate is the greatest usually used precipitant for salting out of proteins.
  3. Ion-exchange chromatography: The dialyzed fraction is applied to a 16 mm x 30 cm column packed with an anion-exchanger, DEAE-cellulose, or DEAE-Sepharose, which is equilibrated beside the dialysis buffer.
  4. Separation by hydrophobic interaction: The protein sample from either of the preceding steps can be applied to a column containing a hydrophobic matrix, namely Phenyl- or Octyl Sepharose, pre-equilibrated with 20 mM Tris-HCl (pH 7.5) comprising thirty percent of saturated (NH4)2SO4.
  5. Affinity chromatography: A sample of affinity chromatography for separation of NAD (P)-binding proteins is the usage of agarose or Sepharose-bound responsive dyes.

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