Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. George Gabriel Stokes named the phenomenon fluorescence in The name was derived from the mineral fluorite (calcium difluoride)

Emission Excitation Spectrofluorometer Detector monochromator

Microscope and Plate Reader Emission Excitation Dichroic Mirror Detector Filter

Fluorescence has ~1000X greater sensitivity than absorbance –Low Noise (emitted light at right angle vs difference) –Linear Signal Response that spans several orders of magnitude ( ,000 Relative Fluorescence Units, RFU)

Gel Electrophoresis Separation of macromolecules based on size and charge –DNA naturally has negative charge (moves from cathode to anode) –Protein has multiple charged states, SDS coating imparts migrating proteins uniform negative charge –Used as both analytical and preparative tool What are the key factors that influence quality of the gel (speed vs resolution)

“Before GE” In 50s and 60s, biochemists separated macromolecules in viscous solutions by gravity or centrifugal forces (Sucrose, CsCl – Spinco) Important developments: colloidal agarose, cross-linked polyacrylamide Use of electric current where charge- carrying molecules move through electrically neutral porous matrix - SPEED

Electrochemistry – tube gel

In most electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel.

Flat Bed Electrophoresis Used for large DNA fragments Large surface area, thick slab – dissipates heat %Agarose related to pore size TAE, TBE LB, SB Used for large DNA fragments Large surface area, thick slab – dissipates heat %Agarose related to pore size TAE, TBE LB, SB

Preparing and Pouring Gels – Determine pore size Adjust total percentage of acrylamide Vary amount of crosslinker – Remove oxygen from mixture – Initiate polymerization Chemical method Photochemical method PAGE – Polyacrylamide Gel Electrophoresis

Continuous and Discontinuous Buffer Systems

SDS Gel Electrophoresis

Avoid smiling!!!!

Temperature and Electrophoresis Important at every stage of electrophoresis –During Polymerization Exothermic Reaction Gel irregularities Pore size –During Electrophoresis Denaturation of proteins Smile effect Temperature Regulation of Buffers

Troubleshooting Guide for Protein Electrophoresis & Analysis eshooting.pdf

Electrophoresis – New Frontiers

Chromatography Much of chromatography is NOT instrument- based: sample preparation, explore bind/elute conditions, establish detection methods, etc.

Chromatography Instrumenation Hitachi HPLC Amersham Akta P-900 (FPLC)