Exam 2

Today we had our second exam.

Statistics:

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Lab 12 - Polyacrylamide Gel Electrophoreses (PAGEs) of GFP

Today we discussed 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.

Specifically we were supposed to use it to run protein samples and in two ways.

• A native gel, in which the proteins migrate at different rates depending on their size (molecular weight), tertiary structure, and charge.
• A denaturing gel, in which the proteins are denatured with high temperature and kept denatured by SDS contained in the electrophoresis buffer, so their rate of migration through the gel depends exclusively on size.

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. Unfortunately we didn't have the reagent to stain the gels (coomasie), so we didn't actually run the gels, although we did talk about the process.

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Lab 11 - Protein quantitation

Wednesday, February 3, 2010

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Bradford reagent, based on Brilliant Blue G-250) to bind to proteins (causing the dye reagent to change to a different color) and measure its absorbance. The more concentrated the protein it binds, the darker the blue resultant color, and the greater the absorbance at 595 nm.

Two relative standard proteins are used, bovine serum albumin (BSA)and bovine gamma-globulin (BGG), to generate absorbance vs. protein concentration curves and then interpolate the absorbance of problem samples to estimate their concentration. The protein samples obtained from the Hydrophobic Interaction Chromatography (HIC) are used as problem samples.

This method is applied when researchers in proteomics discover a new protein and are trying to gather information about it. In our case, we "discovered" GFP, although we wouldn't have a name yet, had it been a truly newly discovered protein.

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Lecture, Chapter - Gene regulation at the RNA level

Today we covered chapter 11, on gene regulation at the RNA level.

We discussed mechanisms of gene regulation between transcription and translation:

• Rate of degradation of mRNA
• Modification of mRNA
• Control of translation by RNA binding proteins
• Anti-sense RNA
• Ribosome alteration (preferential translation of mRNAs)
• RNA interference (RNAi)
• Micro RNA (miRNA)
• Riboswitches

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