Protein purification

ITESM, Campus Qro.
Genetic Engineering Laboratory
Francisco Javier Ramírez Carbajal A00889696
November 13, 2011
Comparative Proteomics
Objectives
* Purify cytoskeletal proteins (actin andmyosin) from five different fish samples and compare them with electrophoresis.
Introduction
Proteomics is known as “the study of an organism’s complete complement of proteins”, this means the studyof all proteins, proteome, in order to know how they work in and outside the cells and understand the biological processes occurring in the organisms. This is a quite complex task because the numberof proteins in an organism is huge, each gene can produce al least ten times as many proteins, and there are some genes that can code approximately 1,000 proteins.
Comparative proteomics is used toanalyze the difference in the expression of proteins in different organisms or in different phases in an organism. In this experiment we will analyze the expression of two cytoskeletal proteins, actin(allows movement of cells and cellular processes) and myosin (has an important role in muscle contraction and is responsible for actin-based motility), by the use of SDS-PAGE.
Materials
* 5 1.5mlfliptop micro tubes.
* 5 screwcap micro tubes.
* 250µl of Bio-Rad Laemmli sample buffer.
* 5 fish samples.
* 1x TGS electrophoresis buffer.
Equipment
* Mini-PROTEAN 3 cellMethods
SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis), the way in which it work is the next, first the SDS that is a detergent denature the proteins in order to have proteins withonly primary structure, this is done dissolving the hydrophobic molecules and then by the Sulfate the protein is covered with negatives charges that in the Electrophoresis Chamber will make the proteinswill migrate towards the positive pole when the electrical field is placed.
By it side the PAGE will help to separate the proteins by size, if proteins were run in a normal gel all will cross it...