Module 6 Flashcards
Describe protein extraction
Cells, tissues or organisms are broken apart
-Sonication: single cells, vibratory probe that generates heat
-French press: organelles intact -> cell bursts
-Osmotic shock
-Digestion: lysozyme
-Detergents: SDS -> break down membranes
-Homogenization: force
-The crude preparation must go through a series of steps to remove cellular contaminants
How can extracted proteins be stabilized
What are the assay requirements for protein purification
How can protein purification be tracked
What are purification methods for physical properties
What is centrifugation
What is differential precipitation
What is dialysis (de-salting)
What is chromatography
What is column chromatography
What is size exclusion chromatography
What is partition coefficient
-Most proteins will migrate relative to their size, so a column can be used to run size standards and estimate the size of your protein of interest
What are the limitations of size exclusion chromatography
What is ion exchange chromatography
-gradient elution can be done using a simple 2 beaker, tube and stirbar setup
-As the elution will occur at the top of the coumn first and work downwards, this is a focusing column
What are DNA/RNA spin columns (ion exchange)
What is affinity chromatography
Describe histamine tag (affinity chromatography)
How do you choose which chromatography method to use
