ELECTROPHORESIS Flashcards
- migration of charged solutes or particles in a supporting medium under the influence of electric field
- migration of small ions/molecules
- migration of charged macromolecules in a porous medium (cellulose acetate, paper or agarose)
- generates an _____ = display of protein zones
- ELECTROPHORESIS
- IONTOPHORESIS
- ZONE ELECTROPHORESIS
- ELECTROPHORETOGRAM
Principle of Electrophoresis:
1.______ carries either a positive (+) or negative (-) charge
- aka
2. In an acid solution, an ampholyte receives protons and thereby carries a net (+) charge and migrates
towards the _____
3. In an alkaline solution, an ampholyte gives up protons and thereby carries a net (-) charge and
migrates towards the ______
- Ampholyte
- zwitterion - Cathode
- Anode
Migration depends on:
1. _______ of molecule
2. _______ of molecule
3. _______ of electrical field
4. _______ of the support medium
5. _______ of operation
- Net electrical charge
- Size and shape
- Strength
- Properties
- Temperature
STAINS
1. protein stains (5)
2. isoenzymes stains (1)
3. lipoprotein stains (3)
- protein stains (5)
- AMIDO BLACK
- BROMPHENOL BLUE
- COOMASIE BRILLIANT BLUE
- NIGROSINE
- PONCEAU S - isoenzymes stains (1)
- NITROTETRAZOLEUM BLUE - lipoprotein stains (3)
- FAT RED 7B
- OIL RED O
- SUDAN BLACK B
- electrophoretic method in w/c proteins are separated on the basis of their pI (isoelectric pH)
- Proteins show considerable variation in pI, but pI values fall in the range pH ____ (many having pIs
between pH 4–7)
- ISOELECTRIC FOCUSING
- 3-12
Establishing pH gradients:
1. Mixtures of molecules containing multiple aliphatic amino & carboxylate groups (buffer molecules)
- included directly in ____
2. Derivatives of Acrylamide containing both reactive double bonds & buffering groups
- incorporated in ____ at time of casting
Gel for Isoelectric Focusing:
1. large-pore convective matrices
- Polymerized with an initiator system including _____ for photo-polymerization
Establishing pH gradients:
1. Carrier Ampholytes (Amphoteric electrolytes)
- ISOELECTRIC FOCUSING
2. Acrylamide buffers
- Polyacrylamide Gel
Gel for Isoelectric Focusing:
1. Polyacrylamide Gel
- Riboflavin
HISTORY OF AUTOMATED ANALYZERS
1. ____ = introduction of 1st automated analyzer by Technicon
2. sequential batch analyzer capable of providing single test result on approx. 40 samples /hour
3. Technicon instruments w/c were next developed; With multiple channels (for diff. tests)
- ____ test results simultaneously
- rate of _____ per hour
4. specialty area w/ rapidly developing arsenal of analyzers
- 3 methods
- 1957
- CONTINUOUS FLOW
- Simultaneous Multiple Analyzer
- 6-12
- 360-720 - IMMUNOCHEMISTRY
- Fluorescence Polarization Immunoassay
- Nephelometry
- Chemiluminescent Detection
BASIC APPROACHES OF AUTOMATED ANALYZERS
______: Liquids (reagents, diluents & samples) are pumped through a system of continuous tubing; Samples are introduced in a sequential manner,
- ____ can be used
- uses ____ channels to run multiple tests on each sample
- major drawbacks
CONTINUOUS - FLOW
- Batch analysis
- parallel single channels
- significant carry-over problems & wasteful use of continuously flowing reagents
BASIC APPROACHES OF AUTOMATED ANALYZERS
______: Uses the force generated by centrifugation to transfer & then contain liquids in separate cuvets for
measurement; Capable of running multiple samples, one test at a time, in a batch
- Major advantage
CENTRIFUGAL ANALYSIS
1. Batch analysis
BASIC APPROACHES OF AUTOMATED ANALYZERS
______: Separation of each sample & accompanying reagents in a separate container; Most popular & versatile; Capability of running multiple tests one sample at a time OR multiple samples one test at a time
- 2 advantages
DISCRETE ANALYSIS
1. Random access, stat capabilities
