Experiment 2: Analysis of Human Blood Proteins Using SDS-PAGE Flashcards
SDS-PAGE stands for ________.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
What is the principle of electrophoresis?
A solution of protein molecules is subjected to an electrical charge, enabling the proteins to migrate at a certain rate depending on their size and inherent charge
What is PAGE?
PAGE is a method of electrophoresis in which the matrix where the proteins are run is an inert cross-linked polyacrylamide gel
How does SDS function as an anionic detergent?
1) denature proteins and linearize them into uniform rod shapes
2) impart a negative charge throughout the proteins to mask their inherent charges
What is the purpose of SDS?
the uniform linear shape along with the overall negative charges ensures that the proteins migrate solely based on their molecular weight
How does denaturation occur by addition of SDS?
SDS molecules bind to the internal hydrophobic cores of the proteins and electrostatic repulsions between the bound SDS molecules cause the protein to unfold
What is the purpose of unfolding the proteins?
1) Renders proteins inactive and unsuitable for studies based on function
2) Removes difference in shape as a factor in fractionation
What are the ingredients of the resolving gel?
Acrylamide 1.5M Tris, pH 8.8 SDS Distilled H2O Ammonium persulphate TEMED
What are the ingredients of the stacking gel?
Acrylamide 0.5M Tris, pH 6.8 SDS Distilled H2O Ammonium persulphate TEMED
What technique can be employed to remove air bubbles from the resolving gel before it is set?
Place isopropanol/n-butanol on top of the resolving gel
What is the function of TEMED?
Catalyzes the polymerization of the gel
What can be done to ensure the protein samples do not float to the top and mix with the buffer?
Preparing the protein samples with sucrose or glycerol to make the samples more dense
What is the significance of the running/resolving gel?
Fractionation based on size occurs here due to the smaller pores of the gel in which the larger proteins experience larger frictional resistance thus move slower through the gel matrix
What is the significance of the stacking gel?
Concentrates the proteins
The protocol describes a _________ system.
discontinuous
