Week 13 (Biochemical Methods IV)

Question 1

Separating/analysing molecules by affinity

Answer 1

• Affinity chromatography
• Western blotting
• ELISA

Question 2

Affinity chromatography: stationary phase

Answer 2

Affinity beads

Question 3

Affinity chromatography: mobile phase

Answer 3

Liquid around the beads

Question 4

Affinity chromatography matrix- Ligand on matrix :Item to be purified

Answer 4

(Ligand on matrix :Item to be purified)

Specific peptide sequence:Antibodies

Specific DNA sequence:Transcription factors

ATP:Protein that binds ATP

Nickel column:His-tagged protein

GSH:GST-tagged protein

Maltose:MBP-tagged protein

Question 5

What is affinity chromatography matrix useful for?

Answer 5

• Used very frequently these days for recombinant proteins.
• Molecular biology used to add extra amino acids to the end of your protein of interest to enable easy purification. The tag can often then be cleaved off enzymatically

Question 6

What are the advantages of affinity chromatography?

Answer 6

Easy

Excellent purification

Quick

Question 7

What are the disadvantages of affinity chromatography?

Answer 7

Not possible for all proteins, especially those harvested from natural sources

Tags on proteins can affect expression or function

Question 8

blocking- 1st step How do we visualise the protein of interest?

Answer 8

1st step – blocking

Membrane with transferred proteins bound The membrane loves to bind protein, need to ‘block’ all the remaining sites where proteins haven’t bound from the transfer Wash the membrane in 4% milk solution or BSA

Question 9

Primary antibody- 2nd step

Answer 9

2nd step – 1° Antibody Need an antibody specific for your protein of interest Place the membrane in a solution containing your 1° antibody

Question 10

Washing’s- 3rd step

Answer 10

3rd step – washing Any non-specifically bound antibody will be washed off Leaving antibody bound only where it is bound to the specific protein of interest

Question 11

antibodies bind to their antigens with

Answer 11

very high affinity

Question 12

Secondary antibody- 4th step

Answer 12

4th step – 2° Antibody An antibody that recognises the 1° antibody Antigen binding sites - antigen specific Constant region - animal specific ie. all antibodies produced in mice have a common sequence in this region, that differs from antibodies produced in rabbits or goats.

Question 13

Washing (again) - 5th step

Answer 13

5th step – washing Any non-specifically bound antibody will be washed off Leaving antibody bound only where it is bound to the specific protein of interest

Question 14

Developing-6th step

Answer 14

Place the membrane in a solution containing the substrate for the enzyme that is conjugated to the 2° antibody

Enzyme reaction will occur only at position where the 2° antibody is located

Question 15

What happens If alkaline phosphatase enzyme is used in developing?

Answer 15

produces a purple band on the membrane wherever the protein of interest is located

Question 16

What happens if horeseradish peroxidase enzyme is used in developing?

Answer 16

produces light.

Need to expose to film and will get a band on the film wherever the protein of interest is located

Question 17

Use of Western Blotting

Answer 17

Protein visualisation/analysis

Question 18

Advantages of western blotting?

Answer 18

Specificity Sensitivity (even in complex mixtures)

Question 19

Disadvantages of Western blotting?

Answer 19

Not possible to extract the protein, only visualise Time-consuming

Question 20

What is ELISA?

Answer 20

Enzyme-linked immunosorbant assay

Question 21

What can be used to detect & quantify a protein in solution?

Answer 21

antibodies

Question 22

what is Indirect ELISA used for?

Answer 22

For quantitating amount of an antibody in a sample

Question 23

What are the steps in indirect ELISA? *

Answer 23

1. There is an antigen coated well 2.Block with non specific protein 3.Add sample and if it contains antibodies they binds to antigen 4. Wash Add enzyme-linked 2° (secondary) antibody 5. Wash 6.Add enzyme substrate 7. Measure absorbance (typically turns yellow)

Question 24

What Is Sandwich ELISA used for?

Answer 24

For quantitating amount of an antigen in a sample

Question 25

What are the steps in sandwhich ELISA?

Answer 25

1. Antibody coated well 2.Block-with non specific protein 3.Add sample 5. Antigen (If present) binds to antibody 6.Wash 7.Add enzyme-slinked second antibody 8.Wash 9.Add enzyme substrate 10.Measure absorbance

Question 26

What is the main use of ELISA?

Answer 26

Protein detection/quantitation Detection and quantisation of small molecules

Question 27

advantages of ELISA

Answer 27

Specificity Sensitivity (even in complex mixtures) Quantitative

Question 28

Disadvantages of ELISA

Answer 28

Time consuming Average of all the molecules in a solution even if in different states

Question 29

What does transfer involve?

Answer 29

Transfer from SDS page to nitrocellulose or PVDF membrane

Question 30

What is Mass spectrometry?

Answer 30

Precise and sensitive measurement of atomic composition of a molecule without prior knowledge of its identity

Question 31

What can you find out from mass spectrometry?

Answer 31

The mass of a protein can be precisely determined - give info on the identity and chemical state of a protein A protein can be identified from the pattern of peptide fragments formed after treatment with a specific protease A peptide can be sequenced Oligosaccharides can be sequenced Lipid species can be identified

Question 32

What are the main components of mass spectrometry?

Answer 32

Mass spectrometers convert analyte into gaseous charged forms (gas-phase ions) A mass analyzer separates the ions according to size A detector measures the abundance of each ion

Question 33

Forming gas-phase ions Two most commonly used techniques to form gas-phase ions of large biochemicals are:

Answer 33

MALDI (Matrix-assisted laser desorption/ionisation) ESI (Electrospray ionisation)

Question 34

What is MALDI?

Answer 34

Sample is dried down in the presence of a volatile compound (matrix). A laser excites & vaporises the matrix converting the sample into the gas phase. Gaseous collisions cause ionisation.

Question 35

What is ESI?

Answer 35

Sample in solution is passed through an electrically charged nozzle into a chamber of very low pressure, evaporating the solvent and giving the charged gaseous analyte

Question 36

A commonly used mass analyzer is:

Answer 36

TOF Time of flight Gaseous ions are accelerated through an elongated chamber under a fixed electrostatic potential. A smaller ion will move through the chamber faster than a large one of the same charge.

Question 37

Identification of proteins using mass spec

Answer 37

Identify a spot or band in a gel Cut it out Digest with trypsin Run on mass spec. Identify the protein from the pattern of peptide fragments by comparing with a database

Question 38

Peptide sequencing

Answer 38

After going through one mass analyzer (to separate different peptides) Bombard peptide with an inert gas to break it up Enter 2nd mass analyser Identify amino acid sequence Called ‘tandem mass spec.’ or ms/ms

Question 39

Post-translational modifications

Answer 39

Post-translational modifications are critical to the function of many proteins eg. Glycosylation Phosphorylation

Question 40

Phosphorylation will increase the mass of the peptide that is phosphorylated – what can mass spectrometry detect?

Answer 40

amount and position of phosphorylation

Question 41

What would happen if your sample did not contain any antigen?

Answer 41

There would be no second antibody binding therefore no signal would be produced

Question 42

Which test is ELISA commonly used in?

Answer 42

HIV

Question 43

start with an SDS page

Answer 43

Question 44

western transfer

Answer 44