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MCAT biochem > analytic techniques > Flashcards

analytic techniques Flashcards

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MCAT flashcards
1
Q

preparative purifications

A

produce significant quantities of purified product for ise

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2
Q

analytical purifications

A

smaller amount of a protein to identify, quantify

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3
Q

protein purification

A

to extract proteins by lysing the cell

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4
Q

protease inhibitors

A

prevent proteolytic degredation

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5
Q

centrifugation

A

separate particles based on mass and density

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6
Q

salting in

A

increase protein solubility

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7
Q

salting out

A

decrease protein solubility
- protein parcipitates out

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8
Q

mobile phase

A

carries mixture through a solid stationary phase where is interacts with the desired product material

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9
Q

stationary phase

A

solid

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10
Q

paper chromatography

A

stationary: filter paper
mobile: liquid solvent that carries solutes up the filter paper via capillary action

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11
Q

thin layer chromatograph

A

stationary: glass or plastic coated in absorbant silica (polar)
mobile: non polar liquid
- seperates molecules via polarity

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12
Q

advantage of TLC over paper chromatography

A

faster, more precise, versatile

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13
Q

retention factor (Rf)

A

how far a solute moves up the stationary plate
-approximates polarity
distance traveled through stationary plate / max distance traveled through the mobile phase

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14
Q

Rf range

A

between 0 -1

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15
Q

lower Rf

A

associated with more polar compounds

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16
Q

higher rf

A

associated with more non polar compounds

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17
Q

disadvantage of TLC

A

analytical purification only (doesnt purify )

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18
Q

column chromatography

A

stationary phase: stop cock that allows product to flow out
mobile: nonpolar
sequential flow between different columns to purify product

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19
Q

what elutes first in column chromatogrphy

A

protein of interest for isolateion

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20
Q

high performance liquid chromatography

A

high pressure
stationary phase: absorbant material with high pressure
mobile phase: liquid
- faster and more precise seperation of compounds

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21
Q

normal phase liquid chromatography

A

gas chromatography
mobile phase: gas
stationary phase: liquid
- measure volitile compounds
- sample is vaporized and travel at different rates due to polarity and boiling point

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22
Q

what happens to compounds with low boiling points in gas chromatography

A

vaporize first and elute first

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23
Q

size exclusion chromatography

A

column packed with gel beads and larger particles elute first

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24
Q

ion-exchange chromatography

A

selects for molecules with specific charge
- anion exchange and cation exchange

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25
Q

anion exchange chromatography

A

named after the type of ion the column is designed to attract
- anion sticks to stationary phase
- cations are eluted
- the column is covered in positive cahrges

26
Q

cation exchange chromatography

A

positive ions stay in the tube
negative ions are eluted
tube is covered in negative charges

27
Q

what is the column of an anion-exchange coated with

A

positive charges to attract the anions

28
Q

what is the column of a cation-exchange coated with

A

negative charges to attract cations

29
Q

what is eluted in cation exchange

A

anions

30
Q

what is eluted in anion exchange chromatography

A

cations

31
Q

affinity chromatography

A

binding affinity of proteins for specific ligands
stationary phase: contains ligand so sample passes through unwanted proteins

32
Q

bond formation of affinity chromatography

A

noncovalent

33
Q

immunoaffinity chromatography

A

antibody within the column to recover proteins bound to a specific antibody

34
Q

electrophoresis

A

seperate molecules based on their migration in the electric field

35
Q

gel electrophoresis

A

samples loaded onto agarose gel and electric field causes induction of a positive and negative pole

36
Q

anode

A

positive pole

37
Q

cathode

A

negative pole

38
Q

electrolytic cells

A

current is applied to drive an otherwise nonspontaneous reaction
ex: gel electrophoresis
cathode is negative and anode is positive

39
Q

galvanic cell

A

anode is negative and cathode is positive
- spontaneous already

40
Q

criteria for distance traveled on gel electrophroesis

A
  1. charge
  2. size
41
Q

SDS-PAGE

A

anionic detergent that gives proteins on a gel electrophoresis UNIFORM negative CHARGE so that proteins seperate only based on SIZE

42
Q

what bonds does SDS disrupt

A
  1. non covalent bonds
  2. imparts even distribution of neg charge
  3. denatures
    - does not efefct covalent bonds (disulfide bridges)
43
Q

what bonds can SDS not disrupt

A

disulfide bridges

44
Q

BME

A

disrupts disulfide bridges in sds page

45
Q

what size proteins migrate more quickly in SDS page

A

smaller

46
Q

what are proteins typically stained with in SDS page

A

comassie blue for visualization on the gel

47
Q

isoelectric focusing

A

allows for fine seperation of proteins with different charge states using a pH gradient
- a protein in pH below the pI will migrate down the gel until it reaches its isoelectric point and will stop in the gel

48
Q

spectroscopy

A

quantify amount of protein in a sample based on absorbance of light at a wavelength

49
Q

bert- lambert law

A

A= Ecl
- a is absorbance
- e is constant
c is concentration of solute
l is path length in cm of transmitted light

50
Q

what does a higher absorbance level mean

A

more protein is present

51
Q

what wavelength do proteins absorb the most

A

uv range from 200nm to 400 nm

52
Q

what wavelength do aromatic proteins absorb best

A

around 280 nm

53
Q

what stain binds to all proteins for visualization

A

comassie blue

54
Q

radioactive labeling

A

visualize proteins in an xray image

55
Q

primary antibody

A

antibody that binds specifically to a protein

56
Q

secondary antibody

A

binds to primary antibody and is tagged for visual bands in protein of interest in western blotting

57
Q

what antibody is tagged in the western blotting

A

secondary antibody

58
Q

radioimmunoassay

A

concentration of protein in sample is assesed by measuring extent to which unlabled proteins compete with radioactive labeled antigens for antibody binding sites

59
Q

ELISA (enzyme linked immunosorbent assay)

A

antigens of a sample are attatched to a solid surface normally on a plate
antibodies specific to the antigen of interest are applied to the plate and these bind their protein antigens
- enzyme is covalently linked to an antibody and the enzyme substate is added to the reaction chamber
- enzyme catalyzes color change that can be measured to determine the amount of protein antigen present

60
Q

western blotting

A

using antibodues specific to a protein of interest to be visualized after electrophoresis

61
Q

what is the goal of elisa

A

to quanitfy the amount of antigen via visualizing an enzyme attatched to an antibody of the antigen

MCAT biochem (11 decks)

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