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BIOL 2321 - Cell Bio Lab > Gel Electrophoresis > Flashcards

Gel Electrophoresis Flashcards

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1
Q

In all cells, how are proteins made?

A

DNA makes RNA which makes proteins

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2
Q

What do proteins do for the cell?

A

provide the machinery for cell morphology and activity

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3
Q

What do investigations into cellular structure and function rely on?

A

techniques that isolate and analyze the macromolecules of the cell (DNA, RNA, and proteins)

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4
Q

What technique was used in lab to explore the structure of which protein?

A

gel electrophoresis was used to explore the structure of the antibody protein immunoglobulin G

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5
Q

Give a basic overview of the gel electrophoresis technique

A

used to isolate DNA, RNA, and proteins

a sample is deposited into a gel and an electrical charge is applied to move the molecules in a direction that is dependent on the size, charge, and shape of the molecule

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6
Q

What does the direction of movement of a particular macromolecule on a gel depend on?

A

the size, charge, and shape

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7
Q

T or F: gel electrophoresis is preparative only

A

false, it can be preparative (isolates and purifies) or analytical (the process itself determines the properties of the isolated component)

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8
Q

What technique is used to separate proteins?

A

SDS polyacrylamide gel electrophoresis (SDS PAGE)

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9
Q

What is the objective of SDS PAGE?

A

to separate and isolate proteins in order to determine the proteins’ molecular masses and structure (quaternary or not)

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10
Q

When does a protein have a quaternary structure?

A

if it consists of more than one polypeptide chain

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11
Q

How are component polypeptide chains of a protein stabilized, aligned, and linked?

A

by the non-covalent bonds and/or covalent disulfide bridges of their R groups

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12
Q

What can the noncovalent bonds between polypeptide chain R groups be?

A

hydrophobic interactions or electrostatic interactions

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13
Q

What was the purpose of this lab?

A

to investigate the IgG antibody using SDS PAGE to determine the size and structure

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14
Q

Describe the function of the gel

A

a semi-solid matrix used to support macromolecules during separation

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15
Q

Which gel type was used in our experiment/is most common for separating proteins?

A

polyacrylamide gel

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16
Q

Describe polyacrylamide

A

a solution of acrylamide monomers that combines with a cross linker to polymerize into long chains which results in the formation of a network of molecular-sized pores in the gel

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17
Q

What determines the pore size of the polyacrylamide gel?

A

the total acrylamide monomer concentration and the % by weight of the crosslinker

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18
Q

T or F: proteins are uncharged molecules

A

false, they are charged

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19
Q

What dictates the charge of a protein?

A

the R groups of its amino acids and the environmental pH

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20
Q

Toward which electrode will a negatively charged macromolecule move on a gel?

A

toward the positively charged anode

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21
Q

Toward which electrode will a positively charged macromolecule move on a gel?

A

toward the negative cathode

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22
Q

what is an isoelectric point? What happens to protein migration if the solution is at a pH of its isoelectric point

A

a pH at which a protein has no overall charge

the protein would not move toward either electrode

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23
Q

What charge will a protein have at pH levels above its isoelectric point? which direction will it move?

A

negative

will move toward the positive anode

24
Q

What charge will a protein have at pH levels below its isoelectric point? which direction will it move?

A

positive

will move toward the negative cathode

25
Q

How do proteins move through the gel?

A

in response to an attractive electric charge (from the electrodes)

26
Q

What variables does the speed at which a protein will move depend on?

A

the protein’s size, shape, and charge: mass ratio

27
Q

Why do size and shape affect the rate of movement of a protein?

A

because the gel has pores and acts as a sieve

28
Q

Will larger or smaller molecules move more easily through a polyacrylamide gel?

A

smaller

29
Q

Will more compact proteins move faster or slower through a polyacrylamide gel than more fibrous or elongated ones?

A

more compact move faster

30
Q

How does charge affect the travel speed of a protein?

A

highly charged molecules migrate faster (stronger attraction forces) than less charged molecules

31
Q

T or F: smaller, more compact, highly charged proteins will move more quickly than larger, elongated and less charged ones

A

true

32
Q

As proteins with the same characteristics (which travel at the same speed) migrate through a gel towards an electrode, what forms?

A

they form distinct bands

each band represents a particular protein

33
Q

How are the bands formed during electrophoresis visualized?

A

by staining the gel and visualizing with the GelDoc EZ imager

34
Q

What does SDS stand for?

A

sodium dodecyl sulfate

35
Q

What is SDS?

A

sodium dodecyl sulfate is an amphipathic detergent (common to household cleaning and hygiene products)

36
Q

What is the purpose of treating protein samples with SDS?

A

it eliminates the differences in charges and shape between proteins so that the only distinguishing feature is molecular mass/size

37
Q

How can SDS accomplish its function of eliminating charge and shape of proteins?

A

SDS is negatively charged so it can mask the charge of proteins and disrupt the noncovalent bonds that hold the structures of proteins together

38
Q

What is tertiary protein structure

A

a single polypeptide

39
Q

What is quaternary structure

A

multiple polypeptide chains

40
Q

How many polypeptide chains does a dimer have? a trimer?

A

dimer = 2

trimer = 3

41
Q

What does it mean if a protein is a heterodimer?

A

it means it is composed of 2 different polypeptide chains

42
Q

What does it mean if a protein is a homotetramer?

A

it is composed of 4 identical polypeptide chains

43
Q

How is 3D protein shape/conformation held together and stabilized?

A

by covalent and noncovlaent bonds

44
Q

What holds together the alpha helices or beta sheets of secondary protein structures?

A

hydrogen bonding of the polypeptide backbone

45
Q

What holds together tertiary and quaternary structures?

A

interactions of the R groups

46
Q

What type of bonds can SDS break down? give examples

A

noncovalent bonds

Van der Waals
hydrogen bonds
ionic bonds

47
Q

Why is SDS not sufficient in completely denaturing proteins?

A

because it only breaks down the noncovalent bonds present but there are also covalent bonds (disulfide bridges)

48
Q

T or F: on its own, SDS is successful in denaturing quaternary proteins

A

false! it cannot break down the disulfide bridges

49
Q

How can disulfide bridges be selectively disrupted without disrupting other covalent bonds?

A

the use of beta mercaptoethanol

50
Q

What is beta ME? What is its purpose in SDS PAGE?

A

beta mercaptoethanol is a reducing agent that breaks down disulfide bridges between cysteine peptides and helps fully denature proteins

51
Q

How is the gel plate orientated for polyacrylamide gels (including position of electrodes)? How are the samples loaded?

A

vertically with the negative cathode at the top and the positive anode at the bottom

Samples are loaded to separate wells at the top

52
Q

Which direction do negatively charged samples run on an SDS PAGE?

A

from top to bottom (positive anode –> negative cathode)

53
Q

How are the molecular masses of the samples determined?

A

by running a sample of known proteins with known molecular masses (a protein ladder/standard) to compare the unknowns to

54
Q

What organism’s serum were proteins isolated from to use in the experiment?

A

bovine serum (Bos taurus)

55
Q

Describe the structure of IgG

A

IgG is a 150 kDa tetramer

made of 2 different polypeptide chains held together by disulfide bridges:
light = 25 kDa
heavy = 50 kDa

the 2 dimers are held together by 2 disulfide bridges

56
Q

What other proteins are present in the bovine serum?

A 
albumin
pre-albumin
acid glycoprotein
haptoglobin
macroglobulin
57
Q

What were the 3 samples run on the SDS PAGE?

A
  1. protein ladder
  2. bovine serum treated with SDS only
  3. bovine serum treated with SDS + BME

BIOL 2321 - Cell Bio Lab (10 decks)

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