Protein Analysis

Question 1

How many different types of proteins are present in

a) saliva
b) blood plasma

Answer 1

a) 2290

b) 2698

Question 2

How is protein typically purified?

Answer 2

1) Tissue homogenisation by sonication, blending, pestle and mortar
2) Separisation of the released material by centrifugation
3) Several chromotography steps
4) Confirmation of protein purify by electrophoresis
5) Comfirmation of protein identift by western immunoblotting
6) Confirmation of protein identity by mass-spectrometry

Question 3

What are the 2 types of density bases ultracentrigugation

Answer 3

Velocity sedimentation

Equlibrium sedimentation

Question 4

What is velocity sedimentation?

Answer 4

Subcellular components sediment at different speeds according to their shape and size when layered over dilute solution containing sucrose
After centrifugation the different components can be collected individually by puncturing the plastic centrifuge tube and collecting drops from the bottom

Question 5

What is equilibrium sedimentation?

Answer 5

Subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings
This is due to a sucrose gradient
The bonds can also be collected by putting a hole in the tube

Question 6

What is gel filtration?

Answer 6

Porous beads are used where the pores are big enough for the right protein to enter the beads
Larger molecules flow through the solvent and smaller molecules are ‘retarded’

Question 7

What is column chromatography?

Answer 7

Mixture of proteins are passed through the columns
The columns have different matrixes so proteins can be separated according to charge, hydrophobicity, size or their ability to bind to other molecules

Question 8

What is affinity chromatography?

Answer 8

Takes advantage of biologically important binding interactions on protein surfaces
The substrate is bound to a matrix
It is used to purify proteins

Question 9

Other than proteins what can be separated using affinity chromatography?

Answer 9

Short DNA oligonucleotides of a specific design can be immobilised in this way and used to purify the DNA binding proteins

Question 10

What is ion exchange chromatography?

Answer 10

Increasing salt concentrations are used to compete for the ionic interactions between the proteins and beads
Proteins are then eluted from the column

Question 11

What is used for cation exchange?

Answer 11

Carboxy methyl CM

Question 12

What is used for anion exchange?

Answer 12

diethylaminoethyl (DEAE)

Question 13

Which proteins are collected earliest in the column in ion exchange chromatography?

Answer 13

Proteins with the lowest affinity for the ion exchange resin as it passes directly through the column

Question 14

What does SDS PAGE stand for?

Answer 14

sodium dodecyl sulphate polyacrylamide gel elctrophoresis

Question 15

What is SDS PAGE?

Answer 15

An electric field is applied to a solution so proteins are separated according to their net charge
The solution contains SDS which binds to hydrophobic regions of protein molecules causing them to unfold

Question 16

What is B-mercaptoethanol used for in SDS PAGE?

Answer 16

Breaks S-S linkages in proteins

Question 17

What is two dimensional gel electrophoresis?

Answer 17

Where the proteins are separated by both charge and size

Question 18

When is 2D gel electrophoresis used?

Answer 18

In proteonomics

Question 19

What does separating protein molecules by isoelectric focusing rely on?

Answer 19

A stable pH gradient

Question 20

What happens to a protein at its isoelectric point?

Answer 20

It has no net charge and no longer migrates in the electric field

Question 21

Antobodies in Immunoblotting are coupled to what?

Answer 21

A detectable enzyme or a fluorescent dye

Question 22

What are the steps of Western Blotting concerning the antibodies used?

Answer 22

a) Electrophoresis transfer
b) Incubate with antibodies (AB)
c) Wash off the antibodies
d) Antibody detection
e) Incubate with enzyme linked antibody (AB2)
f) Wash off excess enzyme linked antibodies
g) Add substrate to detect activity
h) Enzyme detection

Question 23

How are secondary antibodies made?

Answer 23

Rabbit or mouse antibodies are injected into a donkey

The donkey antibodies against the other antibodies can then be purified and labelled

Question 24

What are proteins digested with before mass spec?

Answer 24

Trypsin

Question 25

What is 2D radioactive peptide fingerprinting be used for?

Answer 25

Identification of radioactively labelled sites - phosphorylation and palmitoylation

Question 26

What is palmitoylation?

Answer 26

Covalent attachment of fatty acids eg palmitic acid to cysteine residues of proteis typically in the membrane

Question 27

Which amino acids can be phosphorylated and why?

Answer 27

Serine, threonine and tyrosine because they have OH groups

Question 28

What are the four types of post translational modifications that can appear on amino acids?

Answer 28

Hydroxylation
Methylation
Acetylation
Lipid modification eg palmitoylation