Chapter 3 exploring proteins and proteoms

Question 1

The assay in protein purification measures

Answer 1

enzyme activity

Question 2

NADH and NAD? which absorbs light at

Answer 2

340 nm NADH

Question 3

specific activity

Answer 3

ratio of enzyme activity to the amount of protein in the mixture

Question 4

what happens to specific activity when the protein is pure

Answer 4

it stays constant

Question 5

How are homogenats produced

Answer 5

produced by disruptions of cell membranes

Question 6

Salting out

Answer 6

when protein solubility is decreased when the salt concentration increases

Question 7

Dialysis and how it’s used

Answer 7

to separate proteins from small molecules by using semipermeable membranes

Question 8

Gel-filtration chromatography

Answer 8

separation based on size the proteins will emerge first less volume is accessible

Question 9

separation based on charge

Answer 9

ion exchange chromatography

Question 10

negatively and positively charged proteins can be separated by

Answer 10

anion exchange (diethylaminoaethyl)
cation exchange (carboxymethyl)

Question 11

His-tags are added in which technique

Answer 11

affinity chromatography
immobilised nickelI and other metal ions can be used
to elute the protein, excess imidazole is used to displace the histidine-tags

Question 12

polyacylamid gel and why is it a good choice in electrophoresis

Answer 12

formed by the polymerisation of acrylamide with a small amount of cross-linking agent methylenebisacrylamide

Question 13

B-mercaptoethanol

Answer 13

reduces disulfid bindinger

Question 14

in electrophoresis the mobility of most polypeptides is proportional to

Answer 14

the logarithm of the masses

Question 15

what molecules are used in isoelectric focusing

Answer 15

polyampholytes (small multicharged polymers) having many different pI values

Question 16

the ….. the value of s the slower a molecule moves in a centifual field

Answer 16

smaller

Question 17

a ……. dense particle moves more rapidly than a …… particle

Answer 17

more dense
less dense
the buoyant force is smaller for the more dense particle

Question 18

gradient centrifugation

Answer 18

separation of proteins with different sedimental coefficients

Question 19

sedimentation equilibrium is useful

Answer 19

because it allows the determination of the mass of for example multimeres

Question 20

polyclonal why are they useful in protein detection

Answer 20

derived from multiple anti-body producing cell populations. Because protein of low abundance can be bound by multiple antibodies that recognise different antigenic determinants

Question 21

hybridoma cells how and why

Answer 21

by the fusion of antbody producing cells with myeloma cells

to produce many antibodies with desired specificities

Question 22

enzyme linked immunosorbent assays are used to

Answer 22

detect and quantify proteins

Question 23

ELISA what enzymes are used

Answer 23

enzyme linked immunosorbent assays.
horseradish peroxidase
alkaline phosphatase reacts with a colourless substrate to produce a coloured object

Question 24

types of ELISA

Answer 24

indirect used to detect antibodies

sandwich: used to detect antigens

Question 25

primary antibodies

Answer 25

antibodies specific for the protein of interest

Question 26

secondary antibodies

Answer 26

antibodies specific fo the primary antibodies

Question 27

essential components of mass spectrometers

Answer 27

1- ion source
2- mass analyser
3- detector

Question 28

mass analyser

Answer 28

the analyte ions are distinguished based on mass to charge ratio

Question 29

Time of flight mass analyser

Answer 29

ions are accelerated through an elongated chamber under a fixed electrostatic potential

Question 30

how can the protein ions be broken into smaller peptides

Answer 30

by bombardment with atoms of inert gases such as helium or argon

Question 31

tandem mass spectrometery

Answer 31

two mass analysers

Question 32

what molecule is used in Edman degradation

Answer 32

phenyl isothiocyanat

Question 33

edman degradion is limited to …… residues and why?

Answer 33

50 residues

because not all peptides release the amino acid derivative at each step

Question 34

disulfied bond can be reduced by and how are they prevented from reforming the bonds

Answer 34

B-mercaptoethanol
dithiothreitol
by being alkylated with iodoacetate that forms stable s-carboxymethyl derivatives

Question 35

peptide mass fingerprinting

Answer 35

protein cleavage followed by chromatographic separation

Question 36

what molecules are used in solid phase sythesis of peptides

Answer 36

resin binds to carboxyl-terminal preventing it from reactions with other amino-acids

t-Boc (protecting group of the amino terminal) tert-butyloxycarbonyl can removed by trifluoroacetic acid.
t-Boc

DCC used to activate the aminoacid binding to the carboxyl terminal of the incoming amino acid.
(dicyclohexylcarbodiimid)

the whole peptide is released by HF (hydrofluoric acid)

Question 37

why are x-rays good in molecular crystollographic studies

Answer 37

because their wavelength corresponds to the lenght of a covalent bond

Question 38

synchrotron radiation

Answer 38

the acceleration of electrons in circular orbits close to the speed of the light

Question 39

the spinning of a proton generates

Answer 39

a magnetic momentum

Question 40

a particular state in NMR is favoured when

Answer 40

the state is alligned with the applied field

Question 41

what is the reference used in nmr

Answer 41

tetramethylsilane

Question 42

An interaction between two nuclei is proportional to thr

Answer 42

inverse of the sixth power of the distance between them