Lecture 4 Flashcards
Agarose
most commonly used for DNA
Gel material is non-toxic but most common DNA staining agent ethidium bromide is a carcinogen
Polyacrylamide
polymer material that achieves best separation for protein electrophoresis
The monomer acrylamide is known to be carcinogenic
Caution with exposure when casting the gel
Cellulose acetate
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
Native Electrophoresis
Charged particles will move towards oppositely charged plates
Used to separate and identify DNA and proteins
Separation based on charge to mass ratio
PANIC
Positive is Anode
Negative Is Cathode
SDS-PAGE
proteins are denatured by exposure to SDS which is a powerful detergent
DNA Electrophoresis
DNA is negative and the amount of negative charge is proportional to the size of the piece of DNA
Electrophoresis of small DNA fragments separates them by
size because the charge to mass ratio is the same
Smaller fragments tangle less and move through the matrix more quickly
Problem of separating long DNA fragments
the molecules untangle and stretch out like trains
Pulsed Field Electrophoresis (PFGE)
instead of simply applying + and - charges at opposite ends the field, it is pulsed on and off in a leftright direction
has been the accepted standard for characterizing the DNA of pathogens
Reptation
strands to twist and turn and get tangled
The longer the strand, the more tangling, the slower it moves up the gel
Staining/Destaining
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
Proteins often stained with ?
coomassie blue, Then destained with 5% acetic acid
DNA stained with?
ethidium bromide is visualized under a UV light
Voltages in electrophoresis are?
100’s volts
If pH changes…
the charge on protein will change
Densitometry
Technique to measure average absorbance of a stained spot in a gel
A = –log(I/Io)
Whole blood contains?
Red blood cells
fibrinogen (clotting factor)
proteins
minerals
glucose
hormones
Plasma contains?
fibrinogen (clotting factor)
proteins
minerals
glucose
hormones
Plasma is collected in?
blood is collected into tube with anti-coagulant
Serum contains?
proteins
minerals
glucose
hormones
Serum is collected in?
no anti-coagulant in tube
fibrinogen forms clot
liquid left is serum
Albumin
largest fraction
Alpha-1 globulins
includes High-density lipoprotein (HDL)
associated with “good” cholesterol
Alpha-2 globulins
includes haptoglobin, a protein that binds with hemoglobin that leaves RBCs
Beta globulins
includes transferrin (the iron transport protein)
C3 protein of complement
Gamma globulins
mostly antibodies
Inflammation
Albumin decreases in inflammation
Acute Phase Reactant
Protein levels that rise
α1-antitrypsin, Complement proteins, C- reactive protein(CRP), Ceruplasmin, Haptoglobin
the Excitation Monochromator
One to select the color of light the sample is exposed to
the Emission Monochromator
One to select the color of light coming from the sample that goes to detector
Turbidimeter
measures the light that gets through the sample, like absorbance -log(I/Io) is measured
Nephelometer
measures the scattered light
Nephelometry
Nephelometry is a method for the determination of the cloudiness of asolution
The measured forward scattered light indicates the amount of the insoluble particles present insolution
