2. Protein Purification: Sample Prep + separation

Question 1

List the three groups of starting materials and how do we release proteins from them?

Answer 1

1. Serum plasma and urine: they are liquid samples so they require a little pre-treatment.
2. Cells: lysis.
3. Tissues: homogenization before lysis.

Question 2

Explain the five physical lysis methods?

Answer 2

1. Sonication: high frequency sound waves that result in shear forces which lyse cells. They also prevent heating and foaming.
2. Manual grinding: mortar and pestle which grinds cells or tissues, frozen in liquid nitrogen into fine powder.
3. Mechanical disruption: uses handheld devices like blenders and dounce homogenizers.
4. Liquid homogenization: French pressure cells force cells through a small opening under high pressure which results in shear forces that lyse the cells.
5. Rapid freezing and thawing: freezing causes ice crystals to form which disrupts the cell membrane. Thawing then causes the cell to burst.

Question 3

What is enzymatic lysis and give examples of the enzymes?

Answer 3

They disrupt the cell wall of microcrobes and plants.
1,3 glucarase for yeast; lysozyme for gram negative bacteria; cellulase for plants.

Question 4

What does detergent lysis do?

Answer 4

Disrupts the cell membrane of cells.

Question 5

What are the three types of detergents with examples? Which is/are stronger?

Answer 5

1. Ionic detergents like SDS, CTAB.
2. Zwitterionic detergents like CHAPS.
3. Non-ionic detergents like NP-40 and Triton-X.
   Ionic detergents are stronger.

Question 6

Why are detergents important in cell lysis?

Answer 6

1. They solubilize membrane proteins and lipids.
2. They control protein crystallization.
3. They prevent nonspecific binding during affinity purification and immunoassays.
4. They promote electrophoresis of soluble proteins.

Question 7

How does detergent solubilize proteins?

Answer 7

They mimic the lipid bilayer. They have hydrophobic and hydrophilic regions. the hydrophobic region of the detergent binds to the hydrophobic region of proteins, allowing them to enclose proteins into micelles which prevents the precipitation of proteins and allows for solubilization.

Question 8

What do chaotropic agents do?

Answer 8

They disrupt hydrogen and hydrophobic bonds between and within proteins, which breaks native conformation into a random conformation which results in solubilization.

Question 9

Give examples of chaotropic agents and their functions.

Answer 9

1. Urea, which unfolds proteins.
2. Thiourea which increases us the solubilization power of urea. It is also required to prepare it fresh to decrease the chances of conversion to cyanate or thiocyanate which results in carbamylation which alters the molecular weight of proteins.

Question 10

What do reducing agents do?

Answer 10

They break intra- and inter- molecular disulfide bonds in proteins with cysteine residues.

Question 11

Give examples of reducing agents

Answer 11

DTT and beta mercaptoethanol.

Question 12

How do we remove nucleic acids and why is it important?

Answer 12

By using nucleases which digest DNA and RNA. Nucleic acids can become sticky, which makes pipetting difficult.

Question 13

How do we remove cellular debris?

Answer 13

By centrifugation. Cell fragments get pelleted down and the supernatant contains proteins.

Question 14

How do proteases come about in cell lysis?

Answer 14

Proteases are released during lysis when lysosomes are compromised.

Question 15

What are some solutions against proteases?

Answer 15

Handling at low temperatures, storage at -20/-80C, protease inhibitors.

Question 16

Example of proteases and what does it do?

Answer 16

Endopeptidase. They are a range of enzymes that catalyzes the hydrolysis of peptide bonds in the interior of a protein molecule. The hydroxyl group of endopeptidase carries out a nucleophilic attack on the carbonyl group of the peptide bond which cleaves the peptide bond via hydrolysis.

Question 17

Give five examples of protease inhibitors, and what type of proteases do they work against?

Answer 17

1. PMSF: against serine/cysteine proteases.
2. EDTA/EGTA: against metalloprotease
3. Leupeptin: against serine/cysteine proteases.
4. Pepstatin: against aspartyl proteases.
5. Aprotinin: against serine proteases.

Question 18

What are the types of prefractionation and explain them.

Answer 18

1. Immunoaffinity depletion: Abs immobilized on the gel matrix. Load the sample unwanted proteins, bind to the antibody, wanted proteins flow through.
2. Affinity chromatography: the opposite of number one.
3. Subcellular fractionation: enriches sample with certain organelles to investigate certain classes of proteins. Mechanical homogenization of cells then centrifugation and ultracentrifugation to obtain various cellular fractions and organelles.