Electrophoresis Flashcards
What is electrophoresis?
Technique used to separate, identify or purify macromolecules (e.g. proteins and nuclei acids) based on differences in
Size
Charge
Conformation
What does electrophoresis cause?
Movement of charged particles through medium (paper, gel) under the influence of an electric field
Explain the process of electrophoresis
- When charged molecules are placed in an electric field, they migrate(through a stationary solid medium) towards either the positive (anode) or negative(cathode) electrode
- current from one electrode repels molecules away while the other attracts the molecule
- Frictional force of the supporting matrix acts as a “molecular sieve”, separating the molecules
What do electrophoresis gel consist of?
Microscopic pores of a defined size range that act as a molecular sieve
What are options for electrophoresis gels?
Agarose
Polyacrylamide
What are options for supporting matrix?
Paper( cellulose acetate paper)
Gel
- agarose
- polyacrylamide
What is agarose?
A polysaccharide extracted from seaweed and used at concentrations of 0.5 to 3%
How are agarose gels made?
- suspending dry agarose power in aqueous buffer, then boiling the mixture until a clear solution is formed.
- Clear solution(at 55 degrees Celsius) is poured into a casting tray and allowed to cool to room temperature and solidify into a gel
In electrophoresis, only molecules with…
A net charge will migrate through the gel when it is in an electric field
Which travels through the pores faster, small or large molecules?
Small molecules pass through pores more easily
Two molecules with the differing charge but same size and shape are placed in electrophoresis, which moves faster?
The one with more charge
Molecules with same mass and charge it differ in shape are placed in electrophoresis, which moves faster?
The one with a more compact shape, such as a sphere, migrate through the gel more rapidly than those with an elongated shape, such as a rod
What does the rate of migration through the electric field depend on?
- strength of the field(voltage)
- size and shape of the molecules
- relative hydrophobicity of the samples
- concentration of gel
- ionic strength and temperature of the buffer molecules are in
What are the functions of proteins?
- Enzyme catalysis
- metabolic regulation
- binding transport of small molecules
- gene regulation
- immunological defense
Cell structure
What enable proteins to carry out their biological functions?
Charge, shape, size and solubility
What are the functions of proteins?
Enzyme catalysis
Metabolic regulation
Binding and transport of small molecules
Gene regulation
Immunological defense
Cell structure
How does electrophoretic separation of proteins work?
A protein at a negative or positive charge will migrate towards the electrode of opposite charge
What amino acids are due to a polar, negative charge at physiological pH ?
Glutamic acid and aspartic acid
Which proteins give a polar, positive charge at physiological pH
Lysine, arginine and to a lesser extent histidine
Contrast acidic and basic proteins in an acidic pH
Glutamic acid and aspartic acid have little charge
Lysine and argunine have positive charges
Compare acidic and basic proteins when the pH becomes more basic/raises
- Glutamic and aspartic acid become released a proton and become negatively charged
- Lysine and arginine residues become uncharged pH is raised to high values
Proteins exhibit many 3D shapes and complex folding patterns, what is this determined by?
Their amino acid sequence and post transitional processing such as adding prosthetic groups
The precise three dimensional configuration of a protein is critical to….
It’s biological function
What are the general shapes of proteins?
Spherical, elliptical or rod-like
The number and nature of polypeptides in a protein has large effects on…
It’s mass, size and shape
What is the native form of a protein?
The normal, biological active form of a protein
What effect migration rates of a protein?
The amount and sign of a charge, the size and shape of a native protein, all effect it’s electrophoretic migration rates
What would be the purpose of electrophoresis of native proteins?
Useful clinical and immunological analysis of complex biological samples such as serum
Discus albumin as a serum in electrophoresis
By far the most abundant serum protein and has one of the fastest electrophoretic migration rates
Discuss serum as a a substance tested in gel electrophoresis
Native serum in electrophoresis at alkaline pH results in several zones
What are the functions of albumin
Binds and transports many small molecules, including fatty acids and bilirubin
Involved in ismoregulation
Which serum proteins has the slowest migration rates?
Gamma globulins(antibodies)
What proteins can be observed between albumin and gamma globulins?
Transferrin(ion transfer)
Ceruloplasmin(copper transport)
Macroglobulin(protease inhibitor)
Haptoglobin (binding and conservation of hemoglobin)
How can electrophoretic patterns of human serum protein said in diagnosis of diseases?
- Cirrhosis of liver causes decrease in albumin
- Multiple myeloma(cancer of the immune system) and chronic rheumatoid arthritis causes abnormal increases in the gamma globulins
Recall the steps of gel electrophoresis
Remove rubber dams and comb after 20 minutes when gel has set
Insert casting tray and gel into electrophoresis chamber(tank) and fill chamber with buffer-7.8
Load 30ul of each sample into the appropriate well
Cover tank and activate power source(run at 125V for 45 mins)
Stain proteins
