Electrophoresis Flashcards
What is electrophoresis?
The movement of dispersed particles relative to a fluid under the influence of a spatially uniform electric field
What does electrophoresis separate?
Proteins
Nucleic acids
Glucans
What is agarose?
A linear polymer extracted from red seaweed
What can agarose separate?
Nucleic acids and protein complexes
What is acrylamide?
An organic compound with multiple functional groups (amine and ketone)
What can acrylamide separate?
Proteins and nucleic acids
Why does the liquid phase need to be buffered?
To prevent current-induced pH changes and to minimise heating and it also influences how the gel runs
How large are the molecules that agarose gel can separate?
100-500nm
What is the base pair separation range of agarose gel?
50bp-20kbp
What has the higher resolution; agarose gel or acrylamide gel?
Acrylamide
What does PAGE stand for?
Polyacrylamide gel electrophoresis
What are the 3 types of PAGE?
Native PAGE
Blue native PAGE
SDS-PAGE
Describe native PAGE
Separation of acidic proteins by charge/size
Describe blue native PAGE
The addition of Coomassie blue dye
Provides additional charge on proteins
Can cause dissociation of proteins
Describe SDS-PAGE
The protein is denatured with heat/SDS
The SDS is in a gel buffer: Laemmli buffer
The formation of SDS micelles
The charge of the protein is masked
Separated by mass
Some proteins are recalcitrant to denaturation due to disulfides or thermostable proteins
