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BIOL 2421 - Cellular Biochem > 4. protein experimental techniques > Flashcards

4. protein experimental techniques Flashcards

1
Q

what must researchers do before they can determine: 1. which proteins=expressed in a given cell 2. quantity of specific protein in a cell 3. amino acid sequence of a protein 4. molecular weight of pI of a protein

A

before they can determine any of these, they must first isolate and purify their protein of interest from thousands of proteins in a cell

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2
Q

to purify a protein, what do you start with

A

start with a tissue sample or a large collection of bacterial cells

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3
Q

what is the first step of protein purification

A

they need to make a crude extract

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4
Q

how are crude extracts made

A

by breaking open the cells you start with via differential centrifugation

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5
Q

what is differential centrifugation

A

centrifuging your sample at higher speeds each time, which pellets out different cell components after each spin. The product is an impure sample, but all the proteins are of the same type (ie all mitochondrial proteins)

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6
Q

after differential centrifugation (which isolates proteins based on type), what is the next step

A

the crude extract then undergoes fractionation treatments

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7
Q

what is the purpose of fractionation treatments of the crude extract

A

this separates the proteins into different fractions based on a specific property (ie size, net charge, binding affinities, solubility, etc)

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8
Q

what is the result of fractionation of the crude extract

A

a solution with just a few proteins

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9
Q

what is salting out

A

isolation of a protein based on solubility

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10
Q

what is used in salting out

A

ammonium sulfate

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11
Q

describe the salting out process

A

you add salt to precipitate out your proteins. More salt=less soluble proteins become in the solution.

  • additional salt disrupts the bonds between water and protein, making the proteins aggregate
  • aggregates can be removed via low speed centrifugation
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12
Q

what bonds are disrupted during salting out? what is the result of this

A

bonds between water and protein, which makes the proteins aggregate

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13
Q

what process usually follows salting out

A

dialysis

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14
Q

what is dialysis

A

a process that removes salts and other small molecules from the solution

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15
Q

describe the process of dialysis

A

the precipitate containing the protein of interest is put into a semipermeable tube and resuspended into a buffer. Only small molecules leave the tubing, leaving you with little/no salt left

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16
Q

what is the purpose of column chromatography

A

separation of proteins by their properties

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17
Q

name the two phases of column chromatography

A

the stationary and mobile phase

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18
Q

describe the stationary phase of column chromatography

A

it is a solid, porous material (ie beads, gel, resin) inside the column, and it has very specific properties

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19
Q

describe the mobile phase of column chromatography

A

this is your aqueous protein sample

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20
Q

describe the process of column chromatography

A

a solution from a reservoir is constantly flowing over the column and will eventually drip out the bottom of effluent. The sample is placed on top, proteins migrate down

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21
Q

describe protein migration in column chromatography

A

proteins in solution go from the top to bottom of the tube. As they interact with the porous matrix, they’ll be slowed to different degrees based on protein properties

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22
Q

describe the end result of column chromatography

A

you can collect the (now separated) proteins as they exit the column into fractions

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23
Q

what are the consequences of having a bigger column for column chromatography

A

bigger=better separation

bigger=more diffuse protein gets (not as favourable)

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24
Q

at the end of column chromatography, how do you know if your fractions have protein in them

A

see if they can absorb light at 280nm, because proteins have some absorption due to aromatic residues

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25
Q

T or F: column chromatography helps you identify which proteins you have in your sample

A

false; it only helps you isolate proteins based on their properties

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26
Q

what is the purpose of ion exchange chromatography

A

isolation of proteins based on their net electric charge (sign and magnitude)

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27
Q

describe the process of ion exchange chromatography

A

get a column with beads that are either neg or pos charged. Run the sample through, and oppositely charged proteins will bind to the beads, similarly charged proteins will flow through

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28
Q

define cation exchangers

A

these are positively charged proteins that bind to negative beads

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29
Q

define anion exchangers

A

these are negatively charged proteins that bind to positive beads

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30
Q

what is the purpose of size exclusion chromatography

A

separate proteins based on size

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31
Q

what is another name for size exclusion chromatography

A

gel filtration chromatography

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32
Q

why is gel filtration chromatography another name for size exclusion chromatography

A

because the stationary phase is often a porous gel

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33
Q

what is the result of size exclusion chromatography

A

larger proteins will elute first, as they can’t fit into the cavities of the gel. Smaller proteins get stuck in the cavities, so it takes them longer to elute out

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34
Q

T or F: smaller proteins elute out more quickly than larger proteins in size exclusion chromatography

A

false; the smaller ones get stuck in cavities of the gel, so it takes them longer

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35
Q

what is the purpose of affinity chromatography

A

separation of proteins due to ligand binding affinity

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36
Q

what are examples of ligands that may be used in affinity chromatography

A

ATP or cAMP

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37
Q

describe the process of affinity chromatography

A

the beads have a ligand covalently attached, that the protein of interest will bind to. After one pass, unwanted proteins are eluted out, but protein of interest will be stuck to the beads. A wash with a free ligand elutes the desired protein (because free ligand competes with the bound ligand, so protein will bind to free ligand instead and move its way down)

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38
Q

how has column chromatography become more high tech

A
  • high pressure used to push mobile phase through the column instead of waiting for it to drip through
  • automated injectors to place mobile phase
  • automated/highly sensitive detectors for each released fraction (tells you if/what protein came out)
  • entire process is controllable by computer programs
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39
Q

to purify a protein for the first time, what should you keep in mind

A

lots of trial and error is required. You typically use the cheap methods first, and only the expensive ones when you have a small volume to deal with

40
Q

what are 2 useful parameters when talking about enzymes

A

activity and specific activity

41
Q

define activity

A

total units of enzyme in the solution

42
Q

define specific activity

A

the number of enzyme units per mg of total protein (ie proportion of protein of interest vs all other protein in the sample)

43
Q

when do you stop the purification process

A
  • when further steps no longer increase the specific activity (only applicable if protein=enzyme)
  • stop when only a single protein species is detectable on a gel
44
Q

what method is used to visualize proteins as you purify

A

electrophoresis

45
Q

as you purify, what can electrophoresis help you determine

A

critical protein properties such as isoelectric point and molecular weight (by the migration in an electric field)

46
Q

T or F: electrophoresis helps purify proteins

A

false; it is only used as an analytical method. It does not contribute to purification

47
Q

what type of gel is used in electrophoresis

A

polyacrylamide gel

48
Q

describe polyacrylamide gel

A

porous, will slow proteins to varying degrees based on their charge/mass ratio and their shape

49
Q

what is SDS (include full name)

A

sodium dodecyl sulfate (a detergent)

50
Q

why is SDS added in the electrophoresis procedure

A

it will separate a mixture of proteins. Nearly one SDS molecule will bind per amino acid

51
Q

what type of charge does SDS contribute to

A

contributes a large negative charge to the protein

52
Q

T or F: SDS helps denature proteins

A

true

53
Q

when SDS is applied to a protein, what is the only property we must consider when looking at the final gel? why

A

size. With SDS, all will be negative, and all will be unfolded, so size is the only remaining property that could differ between the proteins

54
Q

T or F: smaller proteins migrate faster through gel

A

true

55
Q

what dye is used to stain the proteins after electrophoresis

A

Coomassie blue

56
Q

what is the full name for SDS-PAGE

A

sodium dodecyl sulfate polyacrylamide gel electrophoresis

57
Q

mobility of most polypeptide chains in linearly proportional to ____

A

the log of their mass

58
Q

what can SDS NOT do

A

break disulfide bonds

59
Q

what is the result of SDS not being able to break disulfide bonds

A

it can be hard to determine the total number of polypeptides present

60
Q

name 2 chemicals that can be added to a sample to break disulfide bonds, and thus separate any polypeptides bound by cysteine residues

A

beta-mercaptoethanol

dithiothreitol

61
Q

what process is used to determine the isoelectric point of a protein

A

isoelectric focusing

62
Q

is SDS used in isoelectric focusing?

A

no

63
Q

what is the charge at pI

A

net charge of 0

64
Q

describe the process of isoelectric focusing

A

first we take molecules that either act as acids or bases, and we run them through the gel. Their migration establishes a pH gradient, and then we can run our protein through

65
Q

in isoelectric focusing, when would protein migration stop

A

migration stops when pI is reached (net charge of zero)

66
Q

if a protein is in a region of the gel where the pH is higher than its pI, what charge will the protein have

A

when pH is higher than pI, we have more deprotonation of functional groups, so the net charge is negative

67
Q

what is the purpose of 2D electrophoresis

A

allows you to find a protein’s molecular weight and pI at the same time

68
Q

what is the first dimension of 2D electrophoresis

A

isoelectric focusing

69
Q

what is the second dimension of 2D electrophoresis

A

SDS-PAGE

70
Q

describe the process of 2D electrophoresis

A

use isoelectric focusing, leaving you with a column gel with bands at different pH values depending on their pI values. Place this on SDS polyacrylamide gel, and then run the gel

71
Q

how do you interpret the results of 2D electrophoresis

A

the first dimension shows you decreasing pI, while the gel shows you decreasing weight (think of it like 2 axes on a graph)

72
Q

what are 3 things you can determine with electrophoresis

A
  • how pure your sample is
  • protein’s molecular weight
  • protein’s isoelectric point
73
Q

what is the crude way you can use electrophoresis to purify

A

run the gel, then physically cut the band you want out of the gel and discard the rest

74
Q

what is the full name of ELISA

A

enzyme-linked immunosorbent assay

75
Q

what is ELISA used for

A

used to determine whether a protein is present in a mixture of proteins

76
Q

describe the process of ELISA

A
  • coat the surface of the wells with sample (antigen)
  • block unoccupied sites with nonspecific protein
  • incubate with primary antibody against the specific antigen
  • incubate with secondary antibody
  • formation of coloured product indicates presence of specific antigen
77
Q

T or F: in an ELISA, all colored products will be the same intensity

A

false; the color intensity is proportional to the concentration of the protein of interest in the sample

78
Q

what is mass spectrometry

A

a non-gel based method to measure protein molecular weight, or the change in molecular weight when a cofactor or ligand is bound

79
Q

what does mass spec measure

A

the time it takes of a protein to travel through an electric field from an injection point to a detector

80
Q

what are the 3 mass spec methods

A
  • MALDI MS
  • ESI MS
  • Tandem MS
81
Q

when is it easiest to determine the amino acid sequence of a protein

A

when you know the DNA sequence of the protein

82
Q

how do you determine the amino acid sequence of a protein if the DNA sequence is unknown

A

the purified protein will be sequenced, and a key part of this will be breaking the polypeptide into smaller fragments

83
Q

what is one method of breaking polypeptides into fragments when trying to determine the amino acid sequence

A

edman degradation

84
Q

what is edman degradation

A

a method that cleaves off the N-terminal residue, but leaves all other bonds intact. The identity of that residue can be determined, and the process can be repeated for each next amino acid in line

85
Q

what is the name of the chemical in edman degradation that labels the amino-terminal residue

A

phenylisothiocyanate (PITC)

86
Q

how does edman degradation link to pH

A

in edman degredation, each step alternates between acidic and basic pH

87
Q

what is one method used to break peptide bonds in an isolated and purified protein

A

acid hydrolysis

88
Q

what is the purpose of acid hydrolysis

A

break peptide bonds from an isolated and purified protein

89
Q

after acid hydrolysis, what step(s) can be done (and why)

A

chromatography or spectroscopy can be used to identify what amino acids are present and in what proportions

90
Q

what is the purpose of proteases

A

if you know what amino acids are present in a protein but don’t know the order, you can use proteases to digest the polypeptide into a few fragments

91
Q

how do proteases work

A

they only cleave the N or C terminal to certain amino acids (not the middle)

92
Q

after using proteases, how can you determine the amino acid sequence

A

you can look at where the fragments overlap to determine the order

93
Q

describe the process of Tandem MS

A

hydrolyze a protein w/ protease. Injection into the first MS where proteins separate based on mass + charge. One species is selected to go into a collision cell, where it is broken down a bunch more times at random peptide bonds. Run the second MS

94
Q

what is the result of a Tandem mass spec

A

you get a series of peaks plotted on a graph that measures signal intensity and mass

95
Q

how do you analyze the results of a Tandem MS

A

starting at the right of the graph and moving left, each successive peak has one less amino acid than before. The difference in mass from peak to peak identifies which amino acid was lost in each case

BIOL 2421 - Cellular Biochem (21 decks)

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