Lecture 2 Flashcards
(30 cards)
What are 2 methods of clarification? When should you use them?
Cheese cloth - whole plant or animal tissue, no cultures.
Low speed centrifugation - whole plant or animal tissue, no cultures. Can also pellet cells from cell culture homogenates and major heterogenous masses.
What’s in a suspension of a sample after clarification?
Organelles and cytosol.
How fast and how long do you have to centrifuge homogenate to produce pellet with nuclear fraction?
500g, 10 minutes.
How fast and how long do you have to centrifuge homogenate to produce pellet with mitochondrial fraction?
10,000g, 20 minutes.
How fast and how long do you have to centrifuge homogenate to produce pellet with microsomal fraction?
Also, what’s the supernatant?
100,000g, 1 hour.
Cytosol (soluble proteins).
What is the definition of specific activity?
The amount of your target protein relative to the amount of total protein in the sample.
What are the units of specific activity?
units of activity/mg total protein.
What 2 things do you need to know for specific activity?
Amount of total protein.
Amount/activity of target protein.
What is a method for finding out the amount of total protein?
Bradford dye binding assay.
Define Assay
To perform an examination on a chemical in order to test its purity.
If a protein is catalytically active (enzyme), which type of assay should be used, and define what it is.
Activity assay - monitoring breakdown of substrate or accumulation of product.
If a protein is catalytically inactive (enzyme), which type of assay should be used, and define what it is.
Immunoassay - detection of protein itself rather than its activity.
3 benefits with activity assay?
Rapid, high through-put, cheap.
3 downsides to immunoassay?
Slow, low through-put, expensive.
How is an activity assay performed with alkaline phosphatase?
I don’t know. Have a look at the powerpoint.
2 methods for determining levels of target protein in Immunoassay?
ELISA,
Western Blotting.
Downside with western blotting?
Not really quantitative.
7 columns in purification table?
Purification step, Vol (mL), Total protein (mg), [Protein] (mg/mL), Total activity, Activity (units/mL), Specific activity (units/mg total protein).
How is total activity calculated?
Activity*Volume
How is specific activity calculated?
Total activity/Total protein.
What salt is typically used for Salt precipitation?
(NH4)2SO4.
What 2 conditions enable maximum solubility in salt precipitation?
Most effective solvation of protein (H’s in H20 interact w/ -ve charge surface residues,
O’s interact w/ +ve charge surface residues.)
Hydrophobic regions at surface effectively masked by water.
What 2 things happen when salt is added to the water in salt precipitation?
Anions and cations interact with +ve and -ve surface residues, reducing solvation.
Interacts with water, demising hydrophobic surface regions.
What does the concentration of salt that a particular protein will precipitate out at, depend on?
Characteristic amounts of polar, non polar and hydrophobic regions at surface of protein.
