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Biol 4890 > Powerpoint 1 Chapter 3 > Flashcards

Powerpoint 1 Chapter 3 Flashcards

1
Q

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

A

x-ray crystallography or NMR (and sometimes computationally)

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2
Q

Genome

A

the blueprint for what proteins may be expressed

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3
Q

Proteosome

A

the proteins of the genome that are actually expressed

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4
Q

first step in purification

A

harvest crude protein mixture from whole cell lysates

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5
Q

purification of a protein is based on ? (4)

A

solubility, size, charge, binding affinity

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6
Q

salting out

A

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

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7
Q

dialysis

A

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

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8
Q

Gel-filtration chromatography

A

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.

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9
Q

Ion-exchange chromatography

A

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.

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10
Q

? beads used for eluting positively charged proteins

A

carboxymethyl group

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11
Q

? beads used for eluting negatively charged proteins

A

diethylaminoehtyl

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12
Q

HPLC

A

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.

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13
Q

Gel electrophoresis

A

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.

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14
Q

SDS PAGE

A

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

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15
Q

SDS binding

A

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

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16
Q

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

A

mass and mobility

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17
Q

isoelectric focusing

A

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

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18
Q

isoelectric point

A

pH at which protein charge is zero

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19
Q

two-dimensional electrophoresis

A

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

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20
Q

sedimentation coefficient is largely proportional to ?

A

mass/size

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21
Q

affinity chromatography

A

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

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22
Q

most common technique for purifying native and recombinant proteins

A

affinity purification

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23
Q

interactions used in affinity purification

A

antibody and epitope tag interactions

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24
Q

TAP

A

tendem affinity purification.

25
Q

? blue staining

A

Commassie

26
Q

A protein purification scheme can be quantitatively evaluated using ?

A

SDS-PAGE

27
Q

antigen types (2)

A

purified protein/partial peptide sequence and epitope tags

28
Q

antibody structure

A

two heavy and two lights chains connected by sulfide bonds

29
Q

polyclonal vs monoclonal antibodies

A

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

30
Q

hybridoma cells

A

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

31
Q

western blot

A

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.

32
Q

two types of ELISA antibody/antigen detection

A

indirect and sandwich

33
Q

indirect ELISA

A

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.

34
Q

sandwich ELISA

A

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.

35
Q

common test for HIV uses ?

A

indirect ELISA

36
Q

sandwich ELISA commonly used for detection of ?

A

small amounts of antigen such as hormone

37
Q

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

A

6 M, 100 degrees

38
Q

amino acid mixture is separated by ? chromatography

A

ion-exchange

39
Q

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

A

ninhydrin or fluorescamine

40
Q

Protein sequences are determined using ?

A

Edman degradation

41
Q

Edman degradation

A

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

42
Q

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

A

98%, 50

43
Q

CNBr cleavage site

A

carboxyl side of methionine

44
Q

Trypsin cleavage site

A

carboxyl side of lysine and arginine residues

45
Q

chymotrypsin cleavage site

A

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

46
Q

carboxypeptidase A cleavage site

A

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

47
Q

performic acid function

A

breaks disulfide bonds and alkylates them to prevent reformation

48
Q

locate disulfide crosslinks

A

separation and identification of disulfide containing peptides folowing performic acid oxidation

49
Q

merrifield synthesis of peptides

A

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.

50
Q

x-ray crystallography

A

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

51
Q

Common NMR protein labels

A

15N and 13C

52
Q

NRM line caused by ?

A

transition between spin states

53
Q

energy sepatartion between two spin states increase with ? in NMR

A

magnetic field strength

54
Q

2D NMR reveals ? from the nuclear overhauser effect

A

closeness of protons in 3D structure

55
Q

Three approaches to engineer a more stable T4 polymerase

A

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

56
Q

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

A

remove

57
Q

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

A

insert

58
Q

alpha helix structure

A

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

59
Q

qi article

A

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

Biol 4890 (16 decks)

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