Como Trabalhamos


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In this section we describe basic concepts of the methods used in LaNeurotox, particularly those related to the isolation or purification of proteins, as well as techniques employed to perform physicochemical characterization of proteins and of protein-protein interactions.

All texts and explanations are given at the level of undergraduate students in biological sciences. Methods addressed here include electrophoresis, chromatographies, mass spectrometry and immunochemical assays.

For details of a given technique, please visit the lateral menu. 

The isolation or purification of a protein or peptide is a necessary step previous to the study of its physicochemical characteristics, elucidation of its tridimensional structure and full comprehension of its biological properties

Inicially, strategies for purifying a protein are empirical, that is, based on attempts and errors, and should be designed specifically for each molecule. There is no way ahead of experimental determination which methods will work best for a particular protein.

protein purification

Methods for protein purification can be classified into four categories, based either on physicochemical characteristics or on affinity for another molecule.

  1. Size – Mass – Density (ex: centrifugation, dialysts, gel-filtration)
  2. Electric charge (ex: ion exchange chromatography, electrophoresis)
  3. Solubility or hydrophobicity (ex: hydrophobic or reverse phase chromatography)
  4. Affinity Chromatography (explores specific interactions of the molecule with a ligand)

 

fluxogramThe figure represents a typical protocol for protein purification, indicating a number of successive steps, each based on a different method applied to isolate a protein from a complex mixture of biomolecules.

Note the quantity of proteins (100g) present in the starting material and the final amount (0.001g) of purified protein. These numbers are typical for the purification of most proteins, enzymes in particular.

For a successful purification it is essential to have a quantitative method to detect the protein of interest, based on its biological properties, in each of the resulting fractions.   In each step, the protein of interest is separated from the other proteins in the mixture based on a different physicochemical property. As a consequence, after each fractionation step the protein of interest will be very similar with other proteins still  present in the same fraction. Thus, more powerful techniques able to discriminate small differences will be necessary at each new purification step to remove the contaminating molecules as the protein of interest achieves high degree of purity.