Lecture 4

Question 1

What is immunoprecipitation used for?

Answer 1

To purify protein of interest from the complex cellular lysate

Question 2

How does immunoprecipitation work?

Answer 2

1. Cells are lysed w/ triton-x or dounce homogenizer
2. Centrifuge lysates clear them of insoluble proteins and aggregates (supernatant contains soluble proteins; pellet includes insoluble aggregates)
3. Soluble lysate is incubated w/ primary antibody against the protein you want to purify
4. Centrifuge to isolate beads from lysate
5. Remove supernatant
6. Boil and load on SDS PAGE
7. Transfer to membrane probe w/ protein of interest antibody

Question 3

What is differential centrifugation?

Answer 3

Using centrifugation to separate diff organelles from a cell lysate based on differences in density and sedimentation

Question 4

How does differential centrifugation work?

Answer 4

1. Lyse the cells w/ dounce homogenizer
2. Centrifuge @ 600 g –> nuclei in pellet
3. Centrifuge remaining supernatant @ 15,000 g –> mitochondria, peroxisomes, lysosomes in pellet
4. Centrifuge remaining supernatant @ 100,000 g –> plasma membrane, ER, golgi in pellet

Question 5

How does sucrose density gradient centrifugation work?

Answer 5

1. Microsomes from the 100,000 g pellet placed on top of a sucrose density gradient (Top = 1 M, bottom = 2 M)
2. Centrifuge @ 65,000 g –> microsomes settle at a layer in the sucrose gradient of equal density (golgi on bottom, plasma membrane in middle, ER on top)

Question 6

If a protein is not present in any pellet after differential centrifugation, where does it reside?

Answer 6

Cytosol

Question 7

What are membranes made of?

Answer 7

Lipids

Question 8

How many RBCs are needed to make a monolayer of lipids?

Answer 8

Need 2x amount of RBCs b/c surface area of monolayer is 2x that of the RBCs