Electrophoresis Flashcards
What are proteins?
A class of diversified biomolecules , differ in chemical properties like size, shape, charge, solubility etc.
What are the functions of proteins?
- Enzyme catalysis
- metabolic regulation
- binding and transport of small molecules
- gene regulation
- immunological defense
- cell structure
What is an isoelectric point?
At a certain pH, the molecule an be electrically neutral, I.e. negative and positive charges are equal
At this pH, it is the isoelectric point
Which amino acids have negatively charged residues at physiological pH?
Glutamic acid and aspartic acid
What amino acids have polar positive residues?
A protein’s positive charge at physiological pH is due to lysine, arginine and to a lesser extent, histidine
What is the charge of amino acids at acidic pH?
- At an acidic pH, glutamic acid and aspartic acid residues have little charge, while lysine and arginine both have positive charges
- As the pH of the protein solution is raised, glutamic and aspartic acid release a proton and become negatively charged.
- However, lysine and arginine residues becomes uncharged as the pH is raised to high values
What does amino acid sequence and post translational processing determine?
Proteins exhibit many different three-dimensional shapes and complex folding patterns
Why is the 3 dimensional configurations of proteins important?
It is important to its biological function,
- general shapes for proteins are spherical, elliptical or rod-like
- the molecular weight is a function of the number and type of amino acids in the polypeptide chain
What are native proteins?
Proteins that are in their normal, biologically active forms are called native
What factors affect the migration rates of electrophoresis?
The magnitude of charge, the size and shape of a native protein
What is serum?
Serum consists of many different types of proteins. Gel electrophoresis of native serum proteins at alkaline pH results in several zones
Where is albumin synthesized?
In the liver
What is the purpose of electrophoresis of native proteins ?
Useful in clinical and immunological analysis of complex biological samples, such as serum
What is the serum concentration and half life of albumin?
Serum conc.- 3.5-5.5 g/dL and a half life of 20 days
What is the most abundant serum protein ?
Albumin, it also has one of the fastest electrophoretic migration rates
What are the functions of albumin?
Albumin binds and transports many small molecules, including fatty acids and bilirubin
-it is also involved with osmotic regulation
Which serum proteins with the slowest migration rates?
These are gamma globulins (antibodies)
What is electrophoresis?
A technique used to separate, identify or purify macromolecules (e.g. proteins and nucleic acids) based on differences in:
- size
- charge
- conformation
- movement of charged particles through a medium (paper, gel) under the influence of an electric field
What is the function of the supporting matrix in electrophoresis?
The frictional force of the supporting matrix acts as a “molecular sieve” , separating the molecules
What happens In 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
- the electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules
A supporting matrix can be used for the electrophoresis of macromolecules and could be made up of:
-Paper, i.e. cellulose acetate paper
- Gel
- Agarose or
- Polysaccharide
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 powder 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
Which molecules will migrate through the gel when it’s in an electric field?
Tonly molecules with a net charge
