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Pharmacology Teaching and Research

Research Techniques.

Laboratory techniques are crucial for effective laboratory based research. 

Below are descriptions and presentations regarding various types of research techniques. 

 

Chromatography.

 

Polyacrylamide Gel Electrophoresis

PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.

The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size. To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size.

For this different protein molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or quaternary structure. The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size.

After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder (marker).

 

 

Creating an SDS-PAGE Molecular Weight Standard Curve in Excel.

 

 

 

 

 Gel Filtration

Gel filtration chromatography seprarates proteins, peptides, and oligonucleotides on the basis of size. Molecules move through a bed of porous beads, diffusing into the beads to greater or lesser degrees.

Smaller molecules diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules enter less or not at all and thus move through the bed more quickly.

Both molecular weight and three-dimensional shape contribute to the degree of retention.

Gel Filtration Chromatography may be used for analysis of molecular size, for separations of components in a mixture, or for salt removal or buffer exchange from a preparation of macromolecules.

 

 

Basic and Applied information regarding Gel Filtration in the Gel Filtration Handbook.

Gel Filtration Handbook
Gel Filtration Handbook.pdf (2.55MB)
Gel Filtration Handbook
Gel Filtration Handbook.pdf (2.55MB)