Lecture 7 Protein Purification and Identification

Question 1

what does protein purification do?

Answer 1

It isolates the target protein from other cellular components.

Question 2

Why is Protein Purification Important?

Answer 2

Protein purification helps understand the structure and function of a protein.

Question 3

what is the main challenge in protein purification?

Answer 3

Since, cells contain a complex mixture of proteins, DNA, molecules, and organelles, the challenge is to isolate the desired protein.

Question 4

what are the methods for protein isolation?

Answer 4

Size sorting through gel filtration

Charge sorting (ion exchange)

Shape sorting

Sifting (centrifugation)

Question 5

What are the different methods for breaking open a cell?

Answer 5

Mechanical methods, Osmotic pressure and Chemical methods

Question 6

What is the mechanical method used to break open a cell?

Answer 6

Grinding, sonication, vortexing

Question 7

What is the osmotic pressure method for breaking open a cell?

Answer 7

Placing cells in a hypotonic solution

Question 8

What is the chemical method used to break open a cell?

Answer 8

Detergents or chemicals to dissolve membranes

Question 9

Why is it important to choose the right cell type?

Answer 9

Different cells express different proteins. For example, pancreatic beta cells express insulin.

Question 10

what are the different cellular compartments?

Answer 10

Nucleus, mitochondria, and cytosol

Question 11

what are the methods to isolate cell compartments?

Answer 11

Nuclear fraction, Mitochondrial fraction, Cytosolic fraction

Question 12

what is a method to quantify proteins?

Answer 12

Spectrophotometry

Question 13

what is the equation of Spectrophotometry?

Answer 13

A = ε × c × l

Question 14

what is the purpose of Spectrophotometry?

Answer 14

measures protein absorbance

Question 15

what are the factors that chromatology considers to separate proteins?

Answer 15

based on size, charge, or binding interactions.

Question 16

how does Size Exclusion Chromatography work?

Answer 16

Separates proteins by size using porous beads.

Question 17

how are large and small proteins affected in Size Exclusion Chromatography?

Answer 17

Large proteins pass quickly

small ones get trapped.

Question 18

how does Ion Exchange Chromatography work?

Answer 18

Separates proteins by charge using a charged resin.

Question 19

how does Affinity Chromatography work?

Answer 19

Separates proteins based on specific binding interactions (e.g., lock and key mechanism).

Question 19

what is cation exchange in Ion Exchange Chromatography?

Answer 19

binds positive proteins

Question 20

what is anion exchange in Ion Exchange Chromatography?

Answer 20

binds negative proteins

Question 21

what is an example of Affinity Chromatography?

Answer 21

Nickel NTA column binds histidine residues.

Question 22

how does Dialysis work?

Answer 22

Purifies proteins by removing small molecules through a semipermeable membrane.

Question 22

how does High-Pressure Liquid Chromatography (HPLC) work?

Answer 22

Separates proteins using pressure and their hydrophobicity.

Question 23

what method is used to assess protein purity?

Answer 23

SDS-PAGE

Question 24

what are the breaking and reformings of non-covalent bonds like?

Answer 24

Weak bonds constantly breaking and reforming

Question 25

how does SDS-PAGE work?

Answer 25

separates proteins based on size and charge.

Question 26

what type of chromatology are non-covalent bonds important for?

Answer 26

important for affinity chromatography