Chemistry Instrumentation

Ampholyte
a molecule that is positively or negatively charged on the basis of the pH of the solution.
True or false: a protein is an ampholyte
true
Electrophoresis
the migration of charged solutes or particles within a liquid medium under the influence of an electrical field
Isoelectric Point
the pH at which the molecule has no net charge and will not migrate during electrophoresis
Theory of Electrophoresis: what charges go where?
+ = cathode
– = anode
Theory of Electrophoresis: Rate of Migration
1. net charge of the molecule
2. size/shape of molecule
3.electric field strength
4. properties of supporting medium
5. Temperature
Wick flow
The result of electrophoretic process that generates heat, causing evaporation of solvent from the electropheretic support.
Electrophoretic mobility
the rate of migration per unit field strength
What is the Mobility equation?
Mobility = Q/6r?
EM:
1. ____________ related to size of the molecule,
2. _____________ related to Q (net charge)
1. inversely
2. directly
What are the four components of an electrophoresis system?
1. Two buffer chambers or “buffer plates”
2. electrodes
3. Electrophoresis support
4. Wicks
5. Cover
Electrophoretic Stain: Protein Serum
Amido Black, Coomasie Blue, Ponceaus
Electrophoretic Stain: Isoenzyme Ormazan
Nitrotetrazolium Blue
Electrophoretic Stain: Lipoprotein zones
Fat Red 7B, Oil Red O, Sudan Black B
Electrophoretic Stain: DNA fragments
Ethydium Bromide
Electrophoretic Stain: CSF proteins
Silver Nitrate
Electrophoretic Techniques:

Slab Gel

separation of serum of CSF proteins and DNA/RNA fragments; horizontal or vertical
Electrophoretic Techniques:

Disc

5 zones, 20+ fragments, “discontinuities”
Electrophoretic Techniques:

Isoelectric focusing

separates amphoteric compounds; sharp protein zones
Electrophoretic Techniques:

2D

Used to study families of proteins, searches for genetic or disease
Electrophoretic Techniques:

Southern Blot

used to detect fragments of DNA; uses AGE; used to (1) identify a DNA sequence, (2) determine the presence, position, and number of copies of a genome, and (3) typing DNA
Electrophoretic Techniques:

Northern Blot

RNA; RNA probe used for hybridization
Electrophoretic Techniques:

Western Blot

Protein; used to (1) separate, (2) detect and (3) identify one or more proteins in a complex mixture
Electrophoresis: purposes of a buffer
1. carry current
2. Establish the pH
3. Determine electrical charge of solute
Electrophoresis:
What provides the matrix in which separation takes place?
The support medium
Electrophoresis:
Separation is based on what?
differences in charge to mass
size of molecule
Electrophoresis:
Agarose separates what analytes?
separation of
-Serum, urine, or CSF proteins
-hemoglobin variants
-Isoenzymes
-Lipoproteins
Electrophoresis:
Advantages of Agarose
Lower affinity for proteins, native clarity for drying, free of ionizable groups, preventing it from endosmosis
Endosmosis
Highly hydrated ions in the applied current of the system move toward the electrode of the opposite polarity which causes movement of the solvent
Zone Electrophoresis
Produces zones of proteins
Electrophoretic Techniques:

Microchip

alternative to capillari electrophoresis; DNA separations
Go through the different Electrophoresis Clinical Significances
Extra Electrophoresis Packet
a1 zone
Increases: inc. in a-1 AT
Decreases: Bence Jones protein binding (RARE)
a2 zone
Haptoglobin, a2 macroglobin, HDL
beta zone
Represents:
Transferrin
Beta-Lipoprotein
C3 Complement
Elevated: High LDL, Pregnancy, Elevated C3, Increase in IgA
Gamma Zone
In Order: IgG IgA IgM IgD IgE
Single band : Monoclonal Gammopathy
Monoclonal Gammopathy of Undetermined Significance (MGUS)
condition in which an abnormal protein is in the blood