SDS Fingerprinting Flashcards
(8 cards)
What is gel electrophoresis?
- Utilises a gel matrix and an applied electric field to separate molecules (DNA, RNA or proteins) by their physical or chemical properties (size, shape and electric charge)
- Helps to identify known molecules in mixed samples and estimating their concentration
- Used study a gene/protein function or a disease, in forensics and paternity testing, identify specific animals/plants/microbes in a biological sample and to determine evolutionary relationships in DNA profiling
Basic principle of gel electrophoresis
- Gel matrix acts as molecular sieve so that smaller molecules move through it more quickly than larger molecules
- The degree of sieving can be controlled by the type of gel substance = agarose for large molecules (DNA) and polyacrylamide for smaller molecules (proteins or small nucleic acids)
- By changing the concentration of the cell matrix, the higher percentage of agarose/polyacrylamide, the denser the matrix
- The electrodes at the top and bottom of the gel influence the migration of the molecules through the matrix according to their charge
Principles of SDS page
- a polyacrylamide gel matrix = acrylamide forms linear polymers and bisacrylamide introduces cross links between polyacrylamide chains, thus creating a network of tunnels. Their diameter (e.g. sieving power) is determined by the ratio of acrylamide to bisacrylamide and by the concentration of acrylamide
- samples containing proteins are loaded into wells and when an electric field is applied, protein molecules migrate in a direction and at a speed that reflects their size and net charge
- SDS is used to solubilise proteins and coat them with negative charges so that they migrate towards the anode through the polyacrylamide gel matrix
- Denaturation of proteins is made complete by using heat and a reducing agent that breaks the disulfide bonds in or between proteins. Under these conditions, the proteins migrate at a rate that reflects their molecular weight only
How are proteins denatured back to their primary structure?
- Heat disrupts the hydrophobic interactions and dipole-dipole interactions
- A thiol reducing agent (e.g. DTT dithiolthreitol) disrupts any disulfide bonds
- The anionic detergent, sodium dodecyl sulphate (SDS) solubilises, denatures and coats the protein with negative charges
Properties of SDS detergent
- Has a lipophilic tail that dissolves hydrophobic molecules = ensures the membranes are dissolved and the proteins solubilise
- Has an anionic head group which binds non-covalently to proteins with a stoichiometry of around 1 SDS molecule per amino acid = ensures that all proteins are covered with negative charges regardless of their intrinsic net charge
How are proteins made visible in the gel?
A Bromophenol Blue dye is mixed with protein samples. The dye molecules are negatively charged and are smaller than the proteins expected in samples
Gel is also placed into another dye called Instant Blue which binds specifically to proteins and not to other macromolecules (like DNA or lipids). The dye is washed with water and distinct blue bands will appear on the gel, revealing where the proteins are.
Intensity of the blue bands will reflect the abundance of proteins
How is the standard curve/calibration curve made?
- Measure distance migrated (DM) by each band of the molecular marker lane from the bottom of the well
- Plot Y (log molecular weight in kDa) vs X (DM in mm)
- Draw sigmoid curve through the points
- Draw line of best fit in the middle = calibration curve
What voltage is used for the gel?
200V
