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MCAT biochem > analytic techniques > Flashcards

analytic techniques 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
anion exchange chromatography
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
cation exchange chromatography
positive ions stay in the tube negative ions are eluted tube is covered in negative charges
27
what is the column of an anion-exchange coated with
positive charges to attract the anions
28
what is the column of a cation-exchange coated with
negative charges to attract cations
29
what is eluted in cation exchange
anions
30
what is eluted in anion exchange chromatography
cations
31
affinity chromatography
binding affinity of proteins for specific ligands stationary phase: contains ligand so sample passes through unwanted proteins
32
bond formation of affinity chromatography
noncovalent
33
immunoaffinity chromatography
antibody within the column to recover proteins bound to a specific antibody
34
electrophoresis
seperate molecules based on their migration in the electric field
35
gel electrophoresis
samples loaded onto agarose gel and electric field causes induction of a positive and negative pole
36
anode
positive pole
37
cathode
negative pole
38
electrolytic cells
current is applied to drive an otherwise nonspontaneous reaction ex: gel electrophoresis cathode is negative and anode is positive
39
galvanic cell
anode is negative and cathode is positive - spontaneous already
40
criteria for distance traveled on gel electrophroesis
1. charge 2. size
41
SDS-PAGE
anionic detergent that gives proteins on a gel electrophoresis UNIFORM negative CHARGE so that proteins seperate only based on SIZE
42
what bonds does SDS disrupt
1. non covalent bonds 2. imparts even distribution of neg charge 3. denatures - does not efefct covalent bonds (disulfide bridges)
43
what bonds can SDS not disrupt
disulfide bridges
44
BME
disrupts disulfide bridges in sds page
45
what size proteins migrate more quickly in SDS page
smaller
46
what are proteins typically stained with in SDS page
comassie blue for visualization on the gel
47
isoelectric focusing
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
spectroscopy
quantify amount of protein in a sample based on absorbance of light at a wavelength
49
bert- lambert law
A= Ecl - a is absorbance - e is constant c is concentration of solute l is path length in cm of transmitted light
50
what does a higher absorbance level mean
more protein is present
51
what wavelength do proteins absorb the most
uv range from 200nm to 400 nm
52
what wavelength do aromatic proteins absorb best
around 280 nm
53
what stain binds to all proteins for visualization
comassie blue
54
radioactive labeling
visualize proteins in an xray image
55
primary antibody
antibody that binds specifically to a protein
56
secondary antibody
binds to primary antibody and is tagged for visual bands in protein of interest in western blotting
57
what antibody is tagged in the western blotting
secondary antibody
58
radioimmunoassay
concentration of protein in sample is assesed by measuring extent to which unlabled proteins compete with radioactive labeled antigens for antibody binding sites
59
ELISA (enzyme linked immunosorbent assay)
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
western blotting
using antibodues specific to a protein of interest to be visualized after electrophoresis
61
what is the goal of elisa
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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