Lecture 4

most commonly used for DNA Gel material is non-toxic but most common DNA staining agent ethidium bromide is a carcinogen

polymer material that achieves best separation for protein electrophoresis The monomer acrylamide is known to be carcinogenic Caution with exposure when casting the gel

non-toxic, durable material after dried used in clinical applications for proteins because it is non-toxic a glucose polymer made into an ester of acetic acid

Charged particles will move towards oppositely charged plates Used to separate and identify DNA and proteins Separation based on charge to mass ratio

Positive is Anode Negative Is Cathode

proteins are denatured by exposure to SDS which is a powerful detergent

DNA is negative and the amount of negative charge is proportional to the size of the piece of DNA

size because the charge to mass ratio is the same Smaller fragments tangle less and move through the matrix more quickly

the molecules untangle and stretch out like trains

instead of simply applying + and - charges at opposite ends the field, it is pulsed on and off in a leftright direction has been the accepted standard for characterizing the DNA of pathogens

strands to twist and turn and get tangled The longer the strand, the more tangling, the slower it moves up the gel

Proteins and DNA must be visualized with stains After electrophoresis performed, the gel with bands is exposed to stain that sticks to protein or DNA more than the gel

coomassie blue, Then destained with 5% acetic acid

ethidium bromide is visualized under a UV light

100’s volts

the charge on protein will change

Technique to measure average absorbance of a stained spot in a gel A = –log(I/Io)

Red blood cells fibrinogen (clotting factor) proteins minerals glucose hormones

fibrinogen (clotting factor) proteins minerals glucose hormones

blood is collected into tube with anti-coagulant

proteins minerals glucose hormones

no anti-coagulant in tube fibrinogen forms clot liquid left is serum

largest fraction

includes High-density lipoprotein (HDL) associated with “good” cholesterol