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Module 1: Proteomics

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Proteomics - Lesson Summary

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The Key Contents from this Module are:

Amino acids are the monomers that makeup Proteins. They are any group of organic molecules that consist of a basic amino group (―NH2), an acidic carboxyl group (―COOH), and an organic R group (or side chain) that is unique to each amino acid.

Each molecule contains a central carbon (C) atom, called the α-carbon, to which both an amino and a carboxyl group are attached. The remaining two bonds of the α-carbon atom are generally satisfied by a hydrogen (H) atom and the R group.

Proteomics aims to study:

The extent of protein expression
How enzymatic regulations happen (activation or inactivation)
How protein-protein interactions happen
Where the proteins are localized
What is the structure and function of these proteins.

The steps involved in Proteome Analysis are:

1. Protein Extraction
2. Protein Separation
3. Protein Identification
4. Protein Characterization

Cyanine labelling enables accurate analysis of differential expression of proteins between the samples. It is possible to label three different samples within the same 2D-gel, enabling accurate analysis of differences in protein abundance between samples by preventing gel to gel variation.

Cyanine dyes are used for labelling samples because they yield brighter and more stable fluorescence. Cyanines can advantageously replace conventional dyes such as fluorescein and rhodamines. Cy3 and Cy5 are the most popular, used typically combined for 2 colours detection.

How to perform equilibration

The equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation.

The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis.

Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension.

In the first equilibration, add 130 mM DTT, -6m Urea, 2% SDS, 0.375 M Tris-HCL, pH 8.8, 20% glycerol. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking.