Lecture 22 - Purification, detection and characterization of proteins

Question 1

4 characteristics of proteins that can be used for protein purification

Answer 1

Mass/size/shape
Density
Charge
Binding affinity

Question 2

3 broad methods of protein separation

Answer 2

Centrifugation
Electrophoresis
Chromatography

Question 3

Centrifugation : Where is it done and what is required

Answer 3

In a centrifuge tube. Need liquid medium (aqueous) and protein of interest

Question 4

What force acts on particles during centrifugation and how it is measured

Answer 4

Centrifugal force -> measured in earth’s gravity (1 earth gravity = 1g)

Question 5

What happens in centrifuge tube if particles are denser than suspending medium ? And if less dense

Answer 5

Go to bottom of the tube.

Less dense = go to the top of the tube

Question 6

What happens in centrifuge tube if particles have same density as medium

Answer 6

Stay where they are

Question 7

Name of medium and product in bottom after centrifugation where protein is denser

Answer 7

Supernatant = medium. Pellet = particles accumulation in the bottom

Question 8

For a given centrifugal force and proteins of similar shapes, what influences the rate at which the supernatant is cleared of the particles ?

Answer 8

Size/mass ratio of particles.

Question 9

What unit is used to calculate ‘‘size’’ unit of particles during centrifugation/rate of supernatant being cleared from particle

Answer 9

The Svedberg

Question 10

How Svederg units work

Answer 10

The greater the number, the faster the particle goes down the tube

Question 11

Possible utility of centrifugation method

Answer 11

Differential centrifugation to separate cell constituants : Exemple : Nucleus denser than mitochondria so take off pellets of nuclei first and mitochrondrias left in supernatant

Question 12

Electrophoresis IN FREE SOLUTION : What determines the direction of migration

Answer 12

Net charge

Question 13

Electrophoresis IN FREE SOLUTION : What determines the speed of migration

Answer 13

Net charge/Mass ratio

Question 14

What happens during gel electrophoresis

Answer 14

Migrating molecules are impeded by the gel (and larger ones more than smaller ones) so molecules that have a same net charge/mass ratio but diff. size migrate to diff. places

Question 15

What is SDS and how it is used in electrophoresis

Answer 15

sodium dodecylsulfate : Used to denature sample of proteins so that their shape doesn’t influence their migration rate

Question 16

2 characteristics of SDS (charge and binds what)

Answer 16

Negatively charged and binds hydrophobic residues

Question 17

Something particular about proteins denatured with SDS

Answer 17

All have same charge:mass ratio

Question 18

Proteins denatured with SDS : Migration in free solution

Answer 18

All would have the same eletrophoretic mobility

Question 19

Proteins denatured with SDS : Migration in polyacrylamide gel (SDS PAGE = SDS Polyacrylamide gel)

Answer 19

Gel impedes larger molecules so migration rate is inverely related to protein size (polypeptide length)

Question 20

What is the isoelectric point of a substance/protein

Answer 20

the pH at which it carries no charge (sum of all particles’ charges = 0)

Question 21

How protein charge evolves with pH and why

Answer 21

Lower pH = more positive ( NH2 -> NH3 +)

Higher pH = more negative (COOH -> COO-)

Question 22

What influences isolectric point of a protein

Answer 22

its amino acid composition

Question 23

Principle of isoelectric focusing + separates protein based on what **

Answer 23

pH gradient created using special buffers (ampholytes) immobilized in polyacrylamide gel. Separation based on CHARGE

Question 24

How ampholytes work when subjected to electric field

Answer 24

When subjected to electrical field, create pH gradient, lower pH towards cathode (+) and higher pH towards anode (-)

Question 25

How are proteins incorporated in the medium during isoelectric focusing and where do they migrate

Answer 25

incorporated (mixture of proteins) into gel during formation (of gel). Positively charged proteins go towards anode and negatively charged ones go towards cathode

Question 26

Where proteins stop migrating during isoelectric focusing and why

Answer 26

At the point in the medium where the pH is equal to their isoelectric point. Have charge of 0 so electric field doesn’t do anything

Question 27

Two-dimensional gel electrophoresis on mixture of proteins 2 steps and how you pass from 1st to 2nd

Answer 27

1) Isoelectric focusing - separate by charge
2) SDS PAGE separate by size
From first to 2nd step, just have to apply first gel on top of second one

Question 28

Antibodies principle + why useful for protein separation

Answer 28

Can recognize 1 specific chemical including 1 specific epitope of a protein so can be used to isolate 1 protein in a complex mixture

Question 29

Other name for Western blot and principle

Answer 29

Immunoblot. Recognize individual protein species in a mixture of proteins separated by SDS PAGE

Question 30

First step of immunobloting

Answer 30

Use horizontal electric field to transfer proteins from PAGE on a membrane

Question 31

Second step of immunobloting

Answer 31

Incubate membrane with an antibody and wash excess. If needed (but not necessary) incubate w/ 2nd antibody specific to 1st one and wash again.

Question 32

Western blot : Why use a second antibody specific to the first one

Answer 32

To detect the first one better

Question 33

Third step of immunobloting (how visualization is done)

Answer 33

Chromogenic detection : Check where antibody is/which protein

Question 34

How Western blot (immunoblot) could be used to see where in the body certain proteins are expressed more

Answer 34

SDS PAGE on protein mixtures from different cell types / cells from diff. tissues. Probe a western blot of this PAG with antibodies of proteins of interest to see in which cells they (the proteins) occur more

Question 35

Mass spectrometry goal and what is done ultimately (and what is not done)

Answer 35

High-precision determination of the charge/mass ratio of ionized molecules. Used to analyze but not purify

Question 36

Concept of mass spectrometry 3 steps

Answer 36

1) Produce gas-phased ions in a vacuum
2) Measure acceleration of ions in electric field
3) Acceleration depends on mass/charge (m/z) ratio

Question 37

Produce gas-phased ions in a vacuum : Most common method used and principle

Answer 37

Electrospray : Molecules of interest (peptides) chopped with proteases and put in a needle with a strong voltage across it

Question 38

What spectrometer detects about an ion (2)

Answer 38

Mass/charge ratio and abundance -> Can put these in a graph

Question 39

How mass over charge ratio is measured (what units)

Answer 39

Daltons per electron (or proton) charge unit

Question 40

Principle of MS/MS (Tandem MS) spectroscopy

Answer 40

Take a peptide from a first mass spectroscopy experiment, fragment it with high energy collision in an inert gas chamber and do mass spectroscopy on the fragments

Question 41

What second graph of a Tandem MS can show (3)

Answer 41

1) Mass/charge ratio and 2) abundance of different fragments from original peptide + 3) NUMBER OF FRAGMENTS = NUMEBR OF A.A IN ORIGINAL PEPTIDE

Question 42

Possible use of all mass/charge ratios of fragments from a peptide

Answer 42

All have 5 or 6 figures so computer can try to identify which sequence in genome could give a peptide with fragments having these m/z ratio -> 1 possiblity = peptide is identified !

Question 43

How can original peptide mass be estimated

Answer 43

Given that we estimated the number of a.a in the original peptide and each a.a is approx 100 Da, we multiply estimated number of a.a * 100

Question 44

What are proteomics

Answer 44

Analysis of subcellular organelles using MS and computer to find the proteins in them