lecture 5

Question 1

1) What is an assay and what does it measure?

What is an enzyme unit?

Answer 1

Assays are tests that identify protein, they measure enzyme units which are the amount of enzyme needed to catalyze one micro mol of substrate per minute.

Question 2

2) What are some ways to isolate enzyme?

Answer 2

First need to always blend up the cell and centrifuge it to get pellet.
Methods:
1) salting out: different proteins have specific solubilities for ammonium sulphate, so we keep adding until we get precipitate, remove the supernatant and keep the pellet.
2) Dialysis to remove the solvent from the salt (it works like a buffer)
3) Then we do column chromatography to separate proteins based size, charge or affinity to bind to column.

Question 3

3) Compare and contrast the 3 different types of column chromatography.

Answer 3

1) Gel-exclusion: based on size, big proteins go faster because they slip along to sides of the beads and , smallest ones are slowest because they go through the beads,
2) ion exchange: based on charge, if the column is – charged, the most – charge proteins go through fastest, vice versa
3) affinity chromatography: use ligand, protein of interest binds to ligand, others are washed away, then remove using a specific salt.

Question 4

4) Gel electrophoresis:
What is the main function of it?
How does it work? What must we add SDS?

Answer 4

To determine if our purification of protein was effective
Migration based on charge and size. SDS negates native charge, and gives equal charge to mass also denatures proteins and makes them the same shape.

Question 5

5) Isoelectric focusing and 2D electrophoresis:
- what does it tell us?
- How does it work?
- What do we do after this?

Answer 5

• it tells the pI of proteins
• separates proteins are based on charge, the top of the tube had high pH and is – charged, the proteins with the highest pI stay there and the ones with the lowest pI move down until it no longer has a net negative charge.
• then do SDS page to separate these guys by charge

Question 6

6) Ultracentrifugation:
What does this method do for us? What does it give us?
Which shape works bests for centrifugation?

Answer 6

• separate biomolecules based on mass, density and shape and gives us the Sedimentation coefficient
• globular, small proteins centrifuge much faster.
• the bigger molecular mass, the bigger the sedimentation coefficient

Question 7

8) How does Electrospray Ionization work and why is it important?

Answer 7

• Records peaks for proteins, different charges give different peaks.
• gives a VERY accurate molecular weight