Electrophoresis Flashcards
→ A molecule that contains both acidic & basic groups (amino acid = carboxyl & amine groups)
AMPHOLYTE
→ Rate of migration of a charged solute in an electric field
ELECTROPHORETIC MOBILITY
→ Preferential movement of water in 1 direction through electrophoresis medium due to selective binding of 1 type of charge on the surface of the medium
ENDOSMOSIS
a record that consists of the separated components of a mixture produced by electrophoresis in a supporting medium
ELECTROPHORETOGRAM
: type of electrophoresis limited to migration of small ions
IONTOPHORESIS
: type of electrophoresis usually used for migration of macromolecules
ZONE ELECTROPHORESIS
The movement of electrically charged compounds in a medium resulting to their separations based on their electrical charges when an electric current is applied
Electrophoresis
Macromolecules found in:
-proteins in serum
-urine
-cerebrospinal fluid (CSF)
-erythrocytes and tissue and,
-other biologic body fluids
ELECTRODES:
a. Cathode —- negatively charged
b. Anode —- positively charged
Factors Affecting Rate of Migration
Net electric charge of the molecule
Size and shape of the molecule
Strength of electrical field
Temperature of operation
Properties of Supporting Medium
Components of Electrophoresis
- Driving force (electrical power)
- Support Medium
- Buffer
- Sample
- Detecting System
In electrophoresis, heat is produced when_____ flows through a medium that has_____, resulting in an increase in thermal agitation of the dissolved solute (ions) and leading to a decrease in resistance and an increase in current.
Driving force (Electrical Power)
current; resistance
Supporting Medium
Cellulose acetate
Agarose gel
Polyacrylamide gel
Cellulose is acetylated to form cellulose acetate by treating it with ______
a dry, brittle film composed of about___ air space
homogenous medium with uniform pore size and does not absorb the protein
Cellulose acetate
acetic anhydride; 80%
A highly purified uncharged polysaccharide derived from agar requires small amounts of sample (approximately 2 mL) it does not bind protein and, therefore, migration is not affected
Agarose gel
referred to as ____
layers of gel with different pore sizes are used
separates serum proteins into___ or more fractions rather than the usual five fractions separated by cellulose acetate or agarose
Polyacrylamide gel; PAGE
20
-separates proteins on the basis of surface charge and molecular size, as does polyacrylamide gel.
-not widely used because of technical difficulty in preparing the gel
Starch
o Polysaccharide extracted from sea weed.
o Gel casted horizontally
Agarose
o Non-toxic.
o Separate large molecules
Agarose
o Commonly used for DNA separations.
o Staining can be done before or pouring the gel.
Agarose
o Cross-linked polymer of acrylamide.
o Gel casted vertically.
Polyacrylamide Gel
o Potent neuro-toxic.
o Separate small molecules.
Polyacrylamide Gel
o Used for DNA or protein separations.
o Staining can be done after pouring the gel.
Polyacrylamide Gel
is a molecule whose net charge can be either positive or negative
AMPHOLYTE
If buffer is more acidic than the pI -becomes_____ charged and migrates toward the_____
positively ; cathode
If buffer is more basic than the pI -becomes____ charged and migrates toward the____
negatively
anode
5 Buffers
Barbitone buffer - (around 8.0 pH)
Phosphate buffer -( around 7.0 pH)
Tris - borate - EDTA buffer (TBE)-(pH around 8.0)
Tris - acetate - EDTA buffer (TAE)- (pH around 8.0)
Tris - glycine buffer - (pH more than 8.0)
• serum protein separation,
• poor resolution, weak buffer.
Barbitone buffer - (around 8.0 pH)
• Enzyme separation,
• low buffering capacity.- high conductivity
Phosphate buffer-( around 7.0 pH)
• Nucleic acid Separation
• Good resolution , high buffering capacity , low conductivity.
Tris - borate - EDTA buffer (TBE) -(pH around 8.0)
• Nucleic acid separation
• high resolution, high buffering capacity low conductivity.
Tris - acetate - EDTA buffer (TAE)- (pH around 8.0)
• Protein separation
• high buffering capacity , low conductivity
Tris - glycine buffer - (pH more than 8.0)-
Stains
PROTEINS
• Amido Black
• Coomassie Brilliant blue
• Bromphenol Blue
Stains
DNA
• Ethidium Bromide
• Sybr Green or Sybr Gold
Stains
LIPOPROTEINS
• Sudan Black
HEMOGLOBINS
• Ponceau Red
• Amido Black
•Coomassie Briliant blue
It measures the absorbance of stain- concentration of the dye and protein fraction.
Densitometry
Densitometry
BASIC COMPONENT:
1.light source
2.monochromator
3.movable carriage
4.photodetector
5.read-out device
•α1-fetoprotein
•α-antitrypsin
•HDL
α1-Globulins
•haptoglobin
•ceruloplasmin
•α2-macroglobulin
α2-Globulins
•transferrin
•C-reactive protein
β-Globulins
•immunoglobulins
γ-Globulins
based on refractivity (the ability of the substance to bend light)
Refractometry
when the light passes from one medium into another, the path of the light beam changes direction at the boundary surface if its speed in the second medium is different from that in the first
Refractometry
Parts of the Refractometer
- Polarizing Filter
- Glass Hemicylincer
- Viewing Lens
- Cover
- Light Enters in Back
- Metal Case
homogenous medium with uniform pore size and does not absorb the protein
Cellulose acetate
Agarose gel
A highly purified uncharged_____ derived from agar
polysaccharide
Agarose gel
requires small amounts of sample (approximately____)
2 mL
Supporting medium that has uniform pore size
Cellulose acetate
Supporting medium that has different pore size
Polyacrylamide gel
How many fractions are separated by cellulose or agarose?
5
It scans and quantitate electrophoretic pattern.
Densitometry
-the ratio of the two speed of light
Refractive Index
Black wire
Negative
Red wire
Negative
Contains the buffet/ electrolyte solution
Subcell
Where the sample went the sample is placed (usually near cathode)
Sample well
