Module 3, Lab 11 - PAGEs of GFP

Image from Bio Rad

As a follow up of Thursday's lab, we stained the gels with Coomasie G-250 stain and then air dried them for analysis.

(click on pic for a full size image)

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Module 3, Lab 12 - Small Scale Plasmid DNA Purification of pGLO

In this lab students purified the pGLO plasmid following Promega's Wizard Plus SV Minipreps DNA Purification System®.

Next week we will do a restriction enzyme digestion of the plasmid DNA in order to isolate the GFP gene from the plasmid.

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Module 3, Lab 11 - Native and denaturing polyacrylamide gel electrophoreses (PAGEs) of GFP

Today we ran a new kind of electrophoresis: Polyacrylamide Gel Electrophoresis (PAGE) is a process that uses the same principle of agarose gel electrophoresis, but it uses a polyacrylamide gel, a thinner, more expensive kind of gel that provides a higher resolution than its agarose counterpart.

We specifically ran protein samples in two ways.

• A native gel, in which the proteins, in their native state, migrate at different rates depending on their size (molecular weight), 3D structure, and charge.
• A denaturing gel, in which the proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge and a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which they migrate in the gel depends only to their size (molecular weight) and not its charge or shape.
  

The goal was to estimate the size of the green fluorescent protein (GFP) by comparing its migration through each gel with the migration of a molecular weight ruler (a "protein ladder") loaded onto the same gel.

PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa.

We will be able to compare teh results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).

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Exam 2

Statistics for exam 2

Click on pic for full size image

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