Powerpoint 1 Chapter 3

Question 1

3D structure of a purified protein can be determined by ? or ?

Answer 1

x-ray crystallography or NMR (and sometimes computationally)

Question 2

Genome

Answer 2

the blueprint for what proteins may be expressed

Question 3

Proteosome

Answer 3

the proteins of the genome that are actually expressed

Question 4

first step in purification

Answer 4

harvest crude protein mixture from whole cell lysates

Question 5

purification of a protein is based on ? (4)

Answer 5

solubility, size, charge, binding affinity

Question 6

salting out

Answer 6

purification by increasing salt concentration to draw out proteins as most are less soluble at high salt concentrations

Question 7

dialysis

Answer 7

purification using a bag with a semipermeable membrane that allows some molecules out (such as salt) while retain some molecules within (such as proteins

Question 8

Gel-filtration chromatography

Answer 8

proteins passed through column consisting of porous beads made of insoluble but highly hydrated polymers. Large molecules elute first because smaller volume is accessible to them. Small beads diffuse into beads to a greater degree.

Question 9

Ion-exchange chromatography

Answer 9

Proteins passed through column of negatively or positively charged beads. oppositely charged proteins stick to the beads and elute more slowly. Same charge proteins elute easily.

Question 10

? beads used for eluting positively charged proteins

Answer 10

carboxymethyl group

Question 11

? beads used for eluting negatively charged proteins

Answer 11

diethylaminoehtyl

Question 12

HPLC

Answer 12

enhanced version of column separation techniques that implements a column containing a finer material that requires pressure to be applied for flow to occur. Results in higher resolution and rapid separation.

Question 13

Gel electrophoresis

Answer 13

a gel with opposite charge on each side will cause proteins to flow in direction of opposite charge. speed of flow based primarily on size and secondarily on charge.

Question 14

SDS PAGE

Answer 14

sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sensitive protein separation technique where proteins are separated in denatured form

Question 15

SDS binding

Answer 15

one SDS binds to every two amino acids. Has negative charge making protein charge contribution insignificant.

Question 16

SDS PAGE allows separation based entirely upon mass, giving an inverse relationship between ? and ?

Answer 16

mass and mobility

Question 17

isoelectric focusing

Answer 17

a pH gradient is used to find the isoelectric point of a protein

Question 18

isoelectric point

Answer 18

pH at which protein charge is zero

Question 19

two-dimensional electrophoresis

Answer 19

begins with a 1D electrophoresis followed by another 1D electrophoresis 90 degrees to the first

Question 20

sedimentation coefficient is largely proportional to ?

Answer 20

mass/size

Question 21

affinity chromatography

Answer 21

purifying proteins based on a highly specific interaction between protein of interest and other chemicals

Question 22

most common technique for purifying native and recombinant proteins

Answer 22

affinity purification

Question 23

interactions used in affinity purification

Answer 23

antibody and epitope tag interactions

Question 24

TAP

Answer 24

tendem affinity purification.

Question 25

? blue staining

Answer 25

Commassie

Question 26

A protein purification scheme can be quantitatively evaluated using ?

Answer 26

SDS-PAGE

Question 27

antigen types (2)

Answer 27

purified protein/partial peptide sequence and epitope tags

Question 28

antibody structure

Answer 28

two heavy and two lights chains connected by sulfide bonds

Question 29

polyclonal vs monoclonal antibodies

Answer 29

monoclonal antibodies are all identical and bind to same epitope. Polyclonal antibodies are a heterogenous mixture but also all bind to the same epitope.

Question 30

hybridoma cells

Answer 30

fusion of short lived antibody producing cell with an immortal myeloma cell

Question 31

western blot

Answer 31

transfer SDS-PAGE gel to polymer sheet and add radiolabeled specific antibody. Wash to remove unbound radiolabel. Expose and develop to identify protein that interacted with antibody.

Question 32

two types of ELISA antibody/antigen detection

Answer 32

indirect and sandwich

Question 33

indirect ELISA

Answer 33

coat well with antigen. wash. allow specific antibody to bind. wash. enzyme linked antibody binds to specific antibody. wash. add substrate that is converted to observable product by enzyme. Color formation is proportional to amount of specific antibody.

Question 34

sandwich ELISA

Answer 34

coat well with monoclonal antibody. wash. allow antigen to bind. wash. second monoclonal enzyme bound antibody allowed to bind antigen. wash. add substrate that is converted to observable product by enzyme. Color formation is proportional to amount of specific antibody.

Question 35

common test for HIV uses ?

Answer 35

indirect ELISA

Question 36

sandwich ELISA commonly used for detection of ?

Answer 36

small amounts of antigen such as hormone

Question 37

Peptide bonds are hydrolyzed in ?M HCl at ? degrees C

Answer 37

6 M, 100 degrees

Question 38

amino acid mixture is separated by ? chromatography

Answer 38

ion-exchange

Question 39

free amino groups react readily with ? or ? to produce an intense fluorescence

Answer 39

ninhydrin or fluorescamine

Question 40

Protein sequences are determined using ?

Answer 40

Edman degradation

Question 41

Edman degradation

Answer 41

label binds to one end of amino acid chain. Label removed along with one bound amino acid. Repeat.

Question 42

edman degradation efficiency is about ?%. Reliably determines chains of ? AA's

Answer 42

98%, 50

Question 43

CNBr cleavage site

Answer 43

carboxyl side of methionine

Question 44

Trypsin cleavage site

Answer 44

carboxyl side of lysine and arginine residues

Question 45

chymotrypsin cleavage site

Answer 45

carboxyl side of tyrosine, tryptophan, phenylalanine, leucine and methionine

Question 46

carboxypeptidase A cleavage site

Answer 46

amino side of C-terminal AA (not arginine, lysine, or proline)

Question 47

performic acid function

Answer 47

breaks disulfide bonds and alkylates them to prevent reformation

Question 48

locate disulfide crosslinks

Answer 48

separation and identification of disulfide containing peptides folowing performic acid oxidation

Question 49

merrifield synthesis of peptides

Answer 49

t-boc amino acid reacts with DCC. Amino acid transfers to resin where it is then deprotected by CF3COOH.couple with another protected amino acid + DCC. repeat. release from resin with HF.

Question 50

x-ray crystallography

Answer 50

xray beamed through protein crystal. Detector captures diffracted beams into images.

Question 51

Common NMR protein labels

Answer 51

15N and 13C

Question 52

NRM line caused by ?

Answer 52

transition between spin states

Question 53

energy sepatartion between two spin states increase with ? in NMR

Answer 53

magnetic field strength

Question 54

2D NMR reveals ? from the nuclear overhauser effect

Answer 54

closeness of protons in 3D structure

Question 55

Three approaches to engineer a more stable T4 polymerase

Answer 55

reduce number of conformations of the unfolded state, stabilize the alpha-helices, increase the number of hydrophobic interactions in the core

Question 56

(remove/insert) glycine to increase energy difference between folded and unfolded states

Answer 56

remove

Question 57

(remove/insert) glycine to increase energy difference between folded and unfolded states

Answer 57

insert

Question 58

alpha helix structure

Answer 58

N_H of backbone donates electrons to carbonyl of backbone four AA's downstream in a right handed coil. A net dipole runs from the C terminal to the N terminal

Question 59

qi article

Answer 59

PM protein complexes tagged with HPB tag containing BCCD of arabidopsis which can be captured by streptavidin beads. PM proteins tagged, RPS2, is a membrane associated disease