Chromatography and Protein Analysis

Question 1

List five features by which proteins differ.

Answer 1

Size

Charge

Hydrophobicity

Stability

Isoelectric point (pI)

Question 2

What must generally be performed on a protein, prior to analysis?

Answer 2

Purification

Question 3

Protein biopharmaceuticals, such as insulin, must be ______ for use.

Answer 3

Pure

Question 4

True or false: if pH is greater than pI, the protein will be negatively charged.

Answer 4

True

Question 5

If pH is _______ than pI, protein will be positively charged.

Answer 5

Greater

Question 6

Column chromatography is a _____________ method.

Answer 6

Separation

Question 7

In column chromatography, the stationary phase is usually _________.

Answer 7

Solid

Question 8

The ___________ phase is usually liquid or gas, flowing over the stationary phase.

Answer 8

Mobile

Question 9

____________ takes place based on the material’s interaction with the stationary phase, relative to its interaction with the mobile phase.

Answer 9

Separation

Question 10

Material used determines the ability of the column to withstand ___________.

Answer 10

Pressure

Question 11

Gel filtration is an example of ____________ chromatography.

Answer 11

Partition

Question 12

______________ chromatography includes ion exchange and hydrophobic interactions.

Answer 12

Adsorption

Question 13

What is affinity, in the context of protein separation?

Answer 13

The ability of many biomolecules (particularly proteins) to recognise specific chemical shapes and structures

Question 14

A small molecule or ‘___________ ________’ is immobilised in a solid stationary phase.

Answer 14

Affinity ligand

Question 15

Outline the principle of affinity chromatography.

Answer 15

A small molecule or ‘affinity ligand’ is immobilised in a solid stationary phase. The mobile phase containing the protein of interest is passed over the stationary phase. Interaction between the protein and the ligand causes protein to bind to the column. Impurities are washed out of the column. Elution may be achieved by lowering the pH, or adding a competitor molecule. Less commonly, ionic strength elution, or chaotropic agents (such as urea or guanine hydrochloride), can be used to caused disassociation of the ligand

Question 16

How may elution be achieved?

Answer 16

By lowering the pH, or adding a competitor molecule

Question 17

What is a ligand?

Answer 17

A molecule that binds reversibly to a specific molecule, or group of molecules

Question 18

Why must a ligand have chemically-modifiable groups?

Answer 18

To allow it to attach to the matrix, without destroying its binding activity

Question 19

What is a spacer arm used for, and how does it work?

Answer 19

To improve binding between the ligand and target molecule, by overcoming any effects of steric hindrance

Question 20

What is ligand coupling?

Answer 20

The covalent attachment of a ligand to a suitable pre-activated matric, to create an affinity medium

Question 21

Metals, such as copper, cobalt, and zinc (all divalent) can be immobilised in the solid stationary phase, bound to agarose, in ____________ ________ ___________ chromatography.

Answer 21

Immobilised metal affinity

Question 22

Proteins bind to __________ via histidine, cysteine, and tryptophan.

Answer 22

Metals

Question 23

True or false: best binding is achieved in the presence of acidic conditions, with 0.5-1.0 M NaCl.

Answer 23

False

Question 24

In immobilised metal affinity chromatography, how can elution be achieved?

Answer 24

Via changing pH, addition of EDTA, or adding competitor molecules

Question 25

____________ binds to nickel or other divalent cations, through its nitrogen.

Answer 25

Imidazole

Question 26

Following chromatography, the proteins isolated can be visualised using a technique called sodium ___________ sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

Answer 26

Dodecyl

Question 27

Proteins are studied using vertical gels, made of ________________, which acts like a molecular sieve.

Answer 27

Polyacrylamide

Question 28

An ___________ ________ is applied, and proteins, negatively-charged after SDS treatment, move.

Answer 28

Electrical field

Question 29

_____ is a denaturant that unfolds proteins, linearising them, to prevent shape affecting movement, by disrupting hydrogen bonds.

Answer 29

SDS

Question 30

SDS binds to the unfolded proteins, and gives them an overall _____________ charge, masking the protein’s own charge.

Answer 30

Negative

Question 31

True or false: smaller proteins are run on higher-concentration gel.

Answer 31

True

Question 32

____________ proteins are run on lower-concentration gel.

Answer 32

Larger

Question 33

Describe the procedure of SDS-PAGE.

Answer 33

Loading dye is added to sample

The gel is run, and various buffers can be used

Two different buffers are within the buffer (tris-glycine/HCl at pH 6.8 and pH 8.8). Leading buffer ion (chloride) is in the stacking gel; trailing buffer ion (glycine) is in the upper gel tank

The cast is opened

Washing, staining, then washing again is performed

Visualisation is performed

Question 34

After electrophoresis, _________ bands are resolved.

Answer 34

Discrete

Question 35

Coomassie Brilliant Blue is sensitive to ____ µg of protein.

Answer 35

0.1

Question 36

________ staining is sensitive to 1 ng of protein.

Answer 36

Silver