Lecture 7 Protein Purification and Identification Flashcards

1
Q

what does protein purification do?

A

It isolates the target protein from other cellular components.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Why is Protein Purification Important?

A

helps understand the structure and function of a protein.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is the main challenge in protein purification?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what are the methods for protein isolation? SCSS

A

Size sorting through gel filtration

Charge sorting ion exchange

Shape sorting

Sifting through centrifugation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What are the different methods for breaking open a cell? MOC

A

Mechanical methods, Osmotic pressure and Chemical methods

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the mechanical methods used to break open a cell? GSV

A

Grinding, sonication, vortexing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

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

A

Placing cells in a hypotonic solution

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

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

A

Detergents or chemicals to dissolve membranes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Why is it important to choose the right cell type and what is an example to help understand this?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what are the different cellular compartments? NMC

A

Nucleus, mitochondria, and cytosol

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are the methods for isolating cell compartments? NMC

A

Nuclear fraction, Mitochondrial fraction, Cytosolic fraction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what is a method to quantify proteins?

A

Spectrophotometry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what is the equation of Spectrophotometry?

A

A = ε × c × l

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is the purpose of Spectrophotometry?

A

measures protein absorbance

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what are the factors that chromatology considers to separate proteins?

A

based on size, charge, or binding interactions.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

how does Size Exclusion Chromatography work? What does it use?

A

Separates proteins by size using porous beads.

17
Q

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

A

Large proteins pass quickly

small ones get trapped

18
Q

how does Ion Exchange Chromatography work? What does it use?

A

Separates proteins by charge using a charged resin.

19
Q

how does Affinity Chromatography work?

A

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

19
Q

what is cation exchange in Ion Exchange Chromatography?

A

binds positive proteins

20
Q

what is anion exchange in Ion Exchange Chromatography?

A

binds negative proteins

21
Q

what is an example of Affinity Chromatography?

A

Nickel NTA column binds histidine residues.

22
Q

how does Dialysis work?

A

Purifies proteins by removing small molecules through a semipermeable membrane.

22
Q

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

A

Separates proteins using pressure and their hydrophobicity.

23
Q

what method is used to assess protein purity?

A

SDS-PAGE

24
Q

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

A

Weak bonds constantly breaking and reforming

25
Q

how does SDS-PAGE work?

A

separates proteins based on size

26
Q

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

A

affinity chromatography