Lecture 14&15: Western Blot, ELISA, PAGE

Question 1

ELISA meaning

Answer 1

• Enzyme linked immunosorbent assay

Question 2

antigen

Answer 2

molecule or protein of interest

Question 3

antibody

Answer 3

protein produced in responses to an antigen by the immune system

Question 4

epitope

Answer 4

region of the antigen the antibody binds to

Question 5

direct ELISA

Answer 5

• Enzyme converts substrate to product
• Ab binds the antigen with enzyme-linked
• Antigen immobilised to plate
• Quick, cheap, but less specific as initial binding not specific
• High background

Question 6

indirect ELISA

Answer 6

• Same specificity issues as direct
• > 1 secondary Ab can bind primary Ab hence better sensitivity
• Longer to run

Question 7

sandwich ELISA

Answer 7

• Improves specificity
• Improved sensitivity due to reduced background noise

Question 8

competitive ELISA

Answer 8

• For small molecules or proteins
• Relies on competition between native and ‘reporter’ antigen
• Sensitivity improvement

Question 9

enzymes in ELISA

Answer 9

• Horse radish peroxidase
• Alkaline phosphatase
• Beta-galactosidase

Question 10

Non-colormetric methods to increase sensitivity:

Answer 10

chemiluminscence
fluorescnece

Question 11

chemiluminscence

Answer 11

o Emission of light as photons resulting from chemical reaction yielding an electronically excited intermediate or product that either luminesces itself or donates its energy to another molecule for the emission (fireflies, crime scenes, glow sticks)

Question 12

fluorescence

Answer 12

o Excitation of energy state, dropping emits light

Question 13

what is 1D page

Answer 13

o 1D-SDS PAGE
o Polyacrylamide gel electrophoresis

Question 14

what is 2d page

Answer 14

o Isoelectric focusing (IEF)
o Uses electrophoretic mobility dependent on pH
o Stop migrating once they reach their isoelectric point

Question 15

process for 2d page

Answer 15

sample prep (lysis buffer and heat)

gel electrophoresis

membrane transfer

immunodetection

Question 16

what are 1d and 2d page used for?

Answer 16

Analysis of specific protein (western blot) or whole proteome by PAGE:

Question 17

staining for general protein analysis

Answer 17

coomassie blue or silver stain

Question 18

coomassie blue

Answer 18

• Binding causes a shift in absorption max of the dye from 465 to 595nm and itt is the increase that is monitored.

Question 19

silver staining

Answer 19

• Silver ions from silver nitrate interact and bind with certain functional groups
  o Carboxylic acid groups: Asp and Glu
  o Imidazole: His
  o Sulfhydryls: Cys
  o Amines: Lys
• Development process similar for photographic film: silver ions reduced to metallic silver, resulting in brown-black colour

Question 20

advantages of comparative ads page analysis

Answer 20

visual comparison, easily presented and protein purified during analysis

Question 21

disadvantages of comparative ads page analysis

Answer 21

may miss low abundance proteins, reproducibility questionable, time consuming

Question 22

Mass spec techniques for protein identification:

Answer 22

• MALDI-ToF MS
• Peptide mass fingerprinting
• Electroppray MS
• HPLC-MS/MS

Question 23

General proteomics analysis protocol:

Answer 23

1. Isolate protein of interest
2. Digest protein into corresponding peptides using trypsin etc.
3. Analyse peptides formed by MS to identify protein

Question 24

Trypsin:

Answer 24

• Common digestive agent
• Digestion enhanced by DTT and iodoacetamide
• 16h at 24 or 37 degrees
• Optimal temp 55

Question 25

Peptide mass fingerprinting/MALDI-ToF MS brief process

Answer 25

- Protein digests analysed - Mass spectra calibrated using trypsin autolysis peaks - m/z values should have high mass accuracy (10ppm or less) - peptide masses submitted to database of theoretical digestions of all proteins and those whose m/z values exactly math are reported

Question 26

Calibration theory: - Accuracy in ppm or Da or %

Answer 26

o 100ppm = 0.01% o 10ppm = 0.001% o 1ppm = 0.0001%

Question 27

MALDI data:

Answer 27

- Relies only on accurate mass of peptide - Need many peaks for an ID - Works with Coomassie blue stained protein

Question 28

HPLC data:

Answer 28

- Relies on nominal mass of peptide and its fragmentation - Need at least 2 peptides matched - Applicable to silver stained proteins

Question 29

Protein quantitation: method

Answer 29

- New HPLC-MS/MS approach using mass labels - Introduce different mass labels in proteome - Mix 2 samples - Digest entire proteome - For each peptide the mass from each sample differs due to the mass label - Comparative height of the two matched peptides reflects their ratio

Question 30

ICAT and SILAC: advantages

Answer 30

automated, gives relative abundance accurately, detect low abundance proteins

Question 31

ICAT and SILAC: disadvantages

Answer 31

complicated spectra (2x peaks), proteins must contain specific amino acids, expensive

Question 32

iTRAQ adv

Answer 32

automated, relative abundance accurately, low abundance proteins detected

Question 33

iTRAQ disadv

Answer 33

expensive, needs suppliers specific MS to do it