Lecture 14 & 15

Question 1

Why are proteins purified?

Answer 1

So the structure and function of them can be studied in isolation (without any other materials interfering)

Question 2

How are proteins purified?

Answer 2

Breaking up the cells, then filtering to find the proteins by differential centrifugation, then by preparative techniques like
1) Salting out
2) Column Chromatography

Question 3

What are the 3 ways cells can be broken up/lysed?

Answer 3

1) Sonication
2) Shearing
3) Mild detergents

Question 4

Explain sonication

Answer 4

a metal rod is placed into a vial and vibrations creates a build up of pressure until the cells burst

Question 5

Explain shearing

Answer 5

Rather cells are squished into a tiny hole or squished against the wall

Question 6

Explain Mild detergents

Answer 6

They lyse the cell by dissolving their hydrophobic layers

Question 7

What is centrifugation?

Answer 7

when materials are spun so hard that gravity sorts it out by density

Question 8

Differential Centrifugation

Answer 8

Used to filter the proteins from lyse cells. Each time the heaviest component goes to the bottom and the supernatant (liquid with the lighter materials) is placed into a separate vial to be centrifuged again. this process is repeated until the protein wanted is the heaviest component

Question 9

Explain salting out

Answer 9

Used to find specific protein within many. The proteins are lest soluble when neutral and in a high salt concentrations. They precipitate out of the solution by clumping up at their isoelectric point. Ammonium sulphate is the most common solution used.

Question 10

what is the difference between analytical and preparative techniques?

Answer 10

preparative techniques are used to filter to find a specific protein. analytical techniques are used to find the structure and properties

Question 11

What is Column Chromatography?

Answer 11

When proteins get filtered based on their physical and chemical properties

Question 12

What are the three types of column chromatography?

Answer 12

1) Ion exchange chromatography
2) Gel (size chromatography)
3) Affinity Chromatography

Question 13

What is ion exchange?

Answer 13

A preparative technique. Protein get separated by charge. Depending on the type of resin, proteins get electrostatically bonded, while others fall through. To obtain the bonded protein, the pH of the solution is changed.

Question 14

What is Gel (size exclusion) chromatography?

Answer 14

A preparative technique. mixture is pumped into the gel matrix that contains many small holes. the smaller protein will get stuck in the holes and take longer to leave while the bigger ones will exit quicker

Question 15

What is affinity chromatography?

Answer 15

A preparative technique. The specific protein binds to the ligand that is covalently bonded to resin. All the other proteins easily washed through as they don’t bond to anything. Then, to release the protein, the solution is pumped both ligands so the protein will bind onto those free ligands and filter through.

Question 16

what is SDS-Page Gel Electrophoresis?

Answer 16

It separates protein by mass. The proteins are surrounded by negatively charged SDS molecules. The top is the gel is negatively charged and the bottom is positively charged. Both are covered with a buffer. The smaller proteins are able to move towards the positive side because the gel is criss crossed so the big ones cans move down.

Question 17

How is molecular weight it estimated?

Answer 17

The larger the portion, the less it moves. The retention factor is the distance migrated by the protein divided by distance go the dye front. (a lot like (TLC).

Question 18

What is a protein ladder?

Answer 18

a markings of the movement of proteins with known molecular weight. used to estimated the weight of unknown proteins

Question 19

what is a 2D electrophoresis?

Answer 19

it uses the isoelectric focusing (overall charge of protein) first then by molecular mass. once they reach their isoelectric point, they become neutral and they don’t move.

Question 20

Why is 2D electrophoresis a good method to separate for MS analysis?

Answer 20

You can analyze multiple proteins at a time and figure out it’s properties

Question 21

What is a proteome?

Answer 21

entire set of proteins from an organism. Secretary protein set= secretome.

Question 22

NMR?

Answer 22

Nuclear Magnetic Resonance shows the direction the atoms spin.
Limits
- limited by size (small only)
- sensitive to local environment

Question 23

X-ray crystallography

Answer 23

exposes the crystals of protein by removing water.
Pros
- more detailed than NMR
- no size limit
Limit
- open to human error
- picture is static, not using for a protein you want to change

Question 24

Antibodies

Answer 24

a protein, also called immunoglobins made by the body in response to a forge in substance, antigen.

Question 25

Parts of a antibody

Answer 25

shaped like a Y, there 4 chains. 2 heavy chain that make the 2 halves of the Y. 2 light chains outline the heads of the Y. 2 Fab domains and one Fc domain.

Question 26

Fab domain

Answer 26

antigen binding domain

Question 27

Fc domain

Answer 27

binding to the receptors of the host cell (crystallizable fragment)

Question 28

Epitope

Answer 28

a specific spot on a antigen that the antibody binds to. It binds due to stereochemical complementary.

Question 29

What is ELIZA?

Answer 29

Enzyme-Linked Immunosorbent Assay- it detects proteins and quantification

Question 30

How does ELIZA work?

Answer 30

1) the well is coated by antigens
2) antibodies that specifically bind to the antigens are placed in the well and bind to the antigens
3) then enzyme looked antibodies bind to the primary antibodies that are bound to the antigen
4) those enzymes will release a colour as a indicator when bonded to a substrate the is put in the well
5) the brightness of the colour quantifies the amount of antibodies present

Question 31

What is Immunoblotting (westen blotting)?

Answer 31

for of proteins detection and quantification

Question 32

How does Immunoglobulin (western blotting) work?

Answer 32

1) First run a SDS electrophoresis
2) Transfer to the filter membrane
3) wash with your primary antibody
4) wash with ur secondary antibody
5) add substrate and it’ll admit colour you want

Question 33

How does enzyme linked- secondary antibody bind to primary antibody?

Answer 33

The enzyme linked secondary antibody binds to the Fc domain of the primary antibody which is the same of every type of antibody and does not change.