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MBB 231 > Lecture 20 - Biochemical Techniques > Flashcards

Lecture 20 - Biochemical Techniques Flashcards

1
Q

what kind of physical/chemical characteristics can be exploited to learn more about molecules(proteins)?

A
  • size
  • charge
  • specificity
  • solubility
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2
Q

why do we want to purify proteins?

A
  • to understand the structure
  • to understand the mechanisms of action
  • to detect protein modifications
  • to identify protein binding partners
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3
Q

def: rupturing the cell to release the internal contents

A

homogenization

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4
Q

what kind of cells need to be broken to get enough protein

A
  • tissue from an organ
  • tissue culture cells
  • yeast cells
  • bacteria cells
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5
Q

what do we want our cell homogenate to have?

A
  • disrupted the plasma membrane
  • released soluble proteins
  • maintain the integrity of organelles
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6
Q

Lysis buffer needs to help break apart the cell, but not all the ___________

A

organelles

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7
Q

what is frequently added to lysis buffers to prevent protein degradation

A

protease inhibitor

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8
Q

what is the disadvantage of using harsh lysis buffers, like amphipathic detergents?

A

they can denature the protein

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9
Q

what are the two common techniques to fractionate a sample?

A
  1. differential centrigfugation
  2. density gradient centrifugation
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10
Q

different __________ __________ separate organelles or macromolecules

A

sedimentation rates

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11
Q

def: separation of cell organelles or other particles of different size and density by their different rates of sedimentation in a centrifugal field

A

differential centrifugation

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12
Q

T or F: differential centrifugation does not yield a pure sample, so further steps are required

A

true

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13
Q

def: a measure of how rapidly the particle sediments when subjected to centrifugation, expressed in terms of Svedberg units

A

sedimentation coefficient

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14
Q

def: more contaminants can be removed using this kind of centrifugation, samples are placed on top of a gradient, centrifugation causes particles to move into discrete zones, based on the density of the gradient and the density of the particle/organelle

A

density gradient centrifugation

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15
Q

dense particles move into the gradient ______ __________ than less dense

A

more rapidly

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16
Q

if you want a specific protein, what do you need to know?

A

you need to know its properties to be able to identify a specific protein

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17
Q

def: a procedure where a mixture of dissolved components is fractioned as it moves through some type of porous matrix (usually in a column)

A

chromatogaphy

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18
Q

differential and density gradient centrifugation give us relatively pure peroxisomes, but if we want to further purify them, what do we do?

A

use in enzyme assays

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19
Q

what are the 3 types of chromatography?

A
  1. ion-exchange chromatography
  2. gel filtration chromatography
  3. affinity chromatography
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20
Q

def: relies on the differences in surface charge of the macromolecule, beads carry a weak (+) or (-) charge, charge proteins interact with beads to varying degrees, and proteins can be eluted using solutions at characteristic pH and salt concentrations

A

ion-exchange chromatography

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21
Q

proteins can be eluted, come out the bottom of the column, by doing what?

A

washing the column with different pH buffer

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22
Q

what happens as the pH is lowered in ion-exchange chromatography?

A
  • negatively charge amino acids become neutral
  • positively charged amino acids become more abundant
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23
Q

def: relies on difference in the size and shape of the macromolecules, beads have different size pores that allow proteins to percolate through them, smaller proteins enter all the pores and take longer to get through the column, larger proteins pass through the column more rapidly

A

gel filtration chromatography

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24
Q

def: relies on highly specific interactions between a ligand/substrate/antibody and the target, beads have a specific molecule attached to their surface and preferentially bind to the protein being purified, proteins are unbound and purified using salt, a change in pH , or mild detergents

A

affinity chromatography

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25
Q

def: when antibodies are used in affinity chromatography

A

immunoprecipitation

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26
Q

T or F: affinity chromatography usually results in near total purification of the desired molecule in a single step

A

true

27
Q

T or F: if you have a catalase specific antibody, you can purify catalase

A

true

28
Q

def: group of techniques that use an electric field to separate charged molecules

A

electrophoreisis

29
Q

what are the most commonly used support media for electrophoresis?

A

polyacrylamide or agarose

30
Q

what is electrophoresis useful for?

A

characterizing a mixture of soluble proteins

31
Q

what does how quickly molecules move during electrophoresis depend on?

A
  • shape
  • charge
  • size
32
Q

what are detergents used for?

A
  • isolate and analyze membrane proteins
  • solubilize, purify, and reconstitute functional membrane protein systems
33
Q

how are detergents able to disrupt the lipid bilayer?

A
  • they are amphipathic
  • disrupt the hydrophobic interactions, dissolving the lipids
34
Q

prior to electrophoresis, insoluble and soluble proteins are usually exposed to high heat and SDS, which _______ _________ and _________ _______ associations

A

protein-protein, protein-lipid

35
Q

how does SDS bind?

A

ratio of 1 SDS molecule per 2 amino acid residues

36
Q

disulfide bonde reducing agents are also often added to the buffer, like:

A
  • beta-mecratoethanol
  • dithiothreitol (DTT)
37
Q

what happens if SDS and DTT are boiled?

A

eliminates the shape and charge component, therefore proteins can only be separated based on their size

38
Q

when proteins are loaded on a polyacrylamide gell and the electric potential is applied, what happens to the negatively charged proteins/SDS?

A

they run toward to positively charged bottom go the gell

39
Q

the ________ the polypeptide the __________ it moves

A

smaller, faster

40
Q

when does the gel stop?

A

when the smallest proteins reach the bottom

41
Q

def: separates proteins in two dimensions, first by charge and then by size

A

two-dimensional SDS-PAGE

42
Q

what can 2D-gels be useful for?

A

resolving subtle differences in various isoforms of proteins

43
Q

def: uses magnetic and electric fields to separate molecules on charge and mass?

A

mass spectrometry

44
Q

what is the first step of mass spec?

A

proteins are digested by a protease to break them into smaller peptides

45
Q

def: proteins are transferred to a membrane and bound to specific antibodies

A

western blotting or immunoblotting

46
Q

what are the two regions on antibodies?

A
  • they have constant regions that are the same for all antibodies
  • variable regions that are identical to each other but unique for each antibody
47
Q

def: represent a mixture of antibodies produced by group of clonal related B cells

A

polyclonal antibodies

48
Q

def: grown from single cells (cell culture)

A

monoclonal antibodies

49
Q

def: fuse a lymphocyte to an immortalized myeloma cell

A

hybridomas

50
Q

what do hybridomas do?

A

can produce a single antibody type and secrete it in to the culture media for collection

51
Q

def: combining the resolving power gel electrophoresis and the specificity of antibodies

A

western blotting (immunoblotting)

52
Q

what happens to solubilized mixtures of proteins?

A
  1. separated by SDS-PAGE
  2. transferred to membrane using electric current
  3. incubated with a primary antibody wash, then add a second antibody
  4. react with a substrate and image
53
Q

def: the entire hereditary information in the form of DNA. This includes both the genes and the non-coding sequences of the DNA

A

genome

54
Q

def: the entire complement of RNA expressed by a genome in a cell tissue, or organism. This will vary depending on the cell’s physiological state

A

transcriptome

55
Q

def: the entire complement of proteins expressed by a genome in a cell, tissue, or organism. This will vary depending on the cell’s physiological state

A

proteome

56
Q

what can the term proteome be used to describe?

A

all the proteins a cell CAN produce OR all the proteins a cell DOES produce

57
Q

T or F: the transcriptome and proteome are larger than the genome in eukaryotes

A

true, because there are more RNA and proteins than genes due to alternate splicing of genes and post translation modifications

58
Q

def: field that merges computer science with biology to address questions about DNA and gene expression

A

bioinformatics

59
Q

def: can be used to see if tow proteins associate in vitro, often done using affinity chromatography

A

co-immunoprecipitaton assays

60
Q

what’s the key difference between hub proteins and non-hub proteins?

A

hub proteins are more likely to be essential for cellular survival

61
Q

hub proteins have:

A
  • several binding interfaces, binding different proteins at the same time
  • a single binding interface, which is capable of binding several different proteins at different times
62
Q

def: in vivo technique to find where a protein binds within the genome of a cell

A

chromatin immunoprecipitation (CHIP)

63
Q

what biochemical techniques can be used to determine protein structure?

A
  • x-ray crystallography
  • nuclear magnetic resonance spectroscopy
  • cryo-electron microscopy

MBB 231 (19 decks)

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