MGD molecular techniques (protein)

Question 1

how do you separate proteins by size?

Answer 1

SDS-PAGE

Question 2

how do you separate proteins by charge?

Answer 2

isoelectric focusing

Question 3

how do you separate proteins by both size and charge?

Answer 3

2D-PAGE (2 dimensions (size & charge)

Question 4

how do you visualise a specific protein?

Answer 4

western blotting

Question 5

how do you analyse protein function?

Answer 5

enzyme assays (determine quality) - all enzymes have specific function

Question 6

how does protein gel electrophoresis work?

Answer 6

separates a protein mixture according to physical properties e.g. size & charge
techniques include: SDS-PAGE, isoelectric focusing, 2D-PAGE

Question 7

how does SDS-PAGE work?

Answer 7

uses a detergent to denature a protein and impart an equal negative charge per unit mass
so proteins move according to molecular weight
often used in combination with western blotting

Question 8

how does isoelectric focusing work?

Answer 8

uses pH gradient in which a protein migrates to find its isoelectric point (pI)
the proteins are stained to become visible at the pI values

Question 9

how does 2D-PAGE work?

Answer 9

uses both pI and molecular weight to sort a mixture of proteins in a sample on gel

Question 10

what is a monoclonal antibody response?

Answer 10

1 type of antibody produced by
1 type of B cell, binding to
1 type of antigen
(all very specific)

Question 11

what is a polyclonal antibody response?

Answer 11

antibodies produced by lots of B cells, affecting many antigens
(seem more generic)

Question 12

typical immune response activates which type of antibody response?

Answer 12

monoclonal B cell (needs to be activated by T helper cells)

Question 13

when is western blotting used?

Answer 13

a technique used to identify the position of a specific protein following SDS-PAGE (separate by size) using monoclonal antibodies
SNOW-DROP (protein) - like southern but for proteins

Question 14

describe the process of western blotting

Answer 14

1. gel transferred to a nitrocellulose membrane
2. primary antibody raised against the protein of interest added
3. secondary labelled antibody raised against primary antibody added
   (protein, 1st antibody attached, 2nd antibody attached to 1st)
4. visualisation

Question 15

what is ELISA used for?

Answer 15

enzyme assay (protein function)
measure concentration of protein in serum e.g. insulin / cortisol

Question 16

describe the process of ELISA

Answer 16

1. immobilise protein on solid surface (antigen coated well)
2. solution containing specific antibody applied to surface
3. Ab bind to protein of interest, other Ab not bound washed away
4. 2nd ENZYME-linked protein applied, binds to AB-AG complex
5. binding of secondary ab measured by substrate (& enzyme interaction) turning into coloured product
   rate of coloured product formation is proportional to amount of specific antibody