Flashcards in Biotech Final - Lab 3 Deck (16)
The electrophoresis of proteins using polyacrylamide gels allows for the separation of proteins into their ______ _______.
What strong anionic detergent is used in SDS-PAGE?
What is used to reduce proteins to their primary structure?
SDS, heat and reducing agents.
How does SDS denature proteins?
By "wrapping around" the polypeptide backbone binding to the protein in a mass ratio of 1.4:1. SDS also disrupts hydrogen bonds, blocks hydrophobic interactions and substantially unfolds proteins.
What does DDT do?
DDT reduces disulphide bonds.
The SDS-denatured and reduced proteins are flexible rods with uniform _____ charge per _____ _____.
Negative charge per unit length
The larger the complex, the _____ the migration in the SDS sieving gel.
The separation ability of an SDS-PAGE gel is contigent on the concentration of ______ and the amount of ______-______ in a gel.
polyacrylamide and cross-linking.
What does bis-acrylamide do?
Provides rigidity and tensile strength to the gel and forms pores, allowing SDS:protein complexes to pass through them.
The size of the pores in an SDS-PAGE gel decrease as?
The ratio of bis-acrylamide: acrylamide increases.
What buffer system did we use?
What is a discontinuous buffer system?
Buffer in the reservoirs and the sample have a different pH and ionic strength than the buffer used to cast the gel.
What does a discontinuous buffer do?
Effectively concentrates the protein into a small volume allowing for increased resolution.
How is the SDS-PAGE gel stained?
Using either Coomassie Blue or Silver staining. Coomassie Brilliant Blue dye forms strong complexes and stoichiometrically binds with proteins, but does not bind tightly to the SDS-PAGE gel.
Proteins separated by SDS-PAGE are fixed using?
methanol or ethanol:glacial acetic acid, then stained using Coomassie Blue.