Gel Electrophoresis Flashcards
In all cells, how are proteins made?
DNA makes RNA which makes proteins
What do proteins do for the cell?
provide the machinery for cell morphology and activity
What do investigations into cellular structure and function rely on?
techniques that isolate and analyze the macromolecules of the cell (DNA, RNA, and proteins)
What technique was used in lab to explore the structure of which protein?
gel electrophoresis was used to explore the structure of the antibody protein immunoglobulin G
Give a basic overview of the gel electrophoresis technique
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
What does the direction of movement of a particular macromolecule on a gel depend on?
the size, charge, and shape
T or F: gel electrophoresis is preparative only
false, it can be preparative (isolates and purifies) or analytical (the process itself determines the properties of the isolated component)
What technique is used to separate proteins?
SDS polyacrylamide gel electrophoresis (SDS PAGE)
What is the objective of SDS PAGE?
to separate and isolate proteins in order to determine the proteins’ molecular masses and structure (quaternary or not)
When does a protein have a quaternary structure?
if it consists of more than one polypeptide chain
How are component polypeptide chains of a protein stabilized, aligned, and linked?
by the non-covalent bonds and/or covalent disulfide bridges of their R groups
What can the noncovalent bonds between polypeptide chain R groups be?
hydrophobic interactions or electrostatic interactions
What was the purpose of this lab?
to investigate the IgG antibody using SDS PAGE to determine the size and structure
Describe the function of the gel
a semi-solid matrix used to support macromolecules during separation
Which gel type was used in our experiment/is most common for separating proteins?
polyacrylamide gel
Describe polyacrylamide
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
What determines the pore size of the polyacrylamide gel?
the total acrylamide monomer concentration and the % by weight of the crosslinker
T or F: proteins are uncharged molecules
false, they are charged
What dictates the charge of a protein?
the R groups of its amino acids and the environmental pH
Toward which electrode will a negatively charged macromolecule move on a gel?
toward the positively charged anode
Toward which electrode will a positively charged macromolecule move on a gel?
toward the negative cathode
what is an isoelectric point? What happens to protein migration if the solution is at a pH of its isoelectric point
a pH at which a protein has no overall charge
the protein would not move toward either electrode
What charge will a protein have at pH levels above its isoelectric point? which direction will it move?
negative
will move toward the positive anode
What charge will a protein have at pH levels below its isoelectric point? which direction will it move?
positive
will move toward the negative cathode
How do proteins move through the gel?
in response to an attractive electric charge (from the electrodes)
What variables does the speed at which a protein will move depend on?
the protein’s size, shape, and charge: mass ratio
Why do size and shape affect the rate of movement of a protein?
because the gel has pores and acts as a sieve
Will larger or smaller molecules move more easily through a polyacrylamide gel?
smaller
Will more compact proteins move faster or slower through a polyacrylamide gel than more fibrous or elongated ones?
more compact move faster
How does charge affect the travel speed of a protein?
highly charged molecules migrate faster (stronger attraction forces) than less charged molecules
T or F: smaller, more compact, highly charged proteins will move more quickly than larger, elongated and less charged ones
true
As proteins with the same characteristics (which travel at the same speed) migrate through a gel towards an electrode, what forms?
they form distinct bands
each band represents a particular protein
How are the bands formed during electrophoresis visualized?
by staining the gel and visualizing with the GelDoc EZ imager
What does SDS stand for?
sodium dodecyl sulfate
What is SDS?
sodium dodecyl sulfate is an amphipathic detergent (common to household cleaning and hygiene products)
What is the purpose of treating protein samples with SDS?
it eliminates the differences in charges and shape between proteins so that the only distinguishing feature is molecular mass/size
How can SDS accomplish its function of eliminating charge and shape of proteins?
SDS is negatively charged so it can mask the charge of proteins and disrupt the noncovalent bonds that hold the structures of proteins together
What is tertiary protein structure
a single polypeptide
What is quaternary structure
multiple polypeptide chains
How many polypeptide chains does a dimer have? a trimer?
dimer = 2
trimer = 3
What does it mean if a protein is a heterodimer?
it means it is composed of 2 different polypeptide chains
What does it mean if a protein is a homotetramer?
it is composed of 4 identical polypeptide chains
How is 3D protein shape/conformation held together and stabilized?
by covalent and noncovlaent bonds
What holds together the alpha helices or beta sheets of secondary protein structures?
hydrogen bonding of the polypeptide backbone
What holds together tertiary and quaternary structures?
interactions of the R groups
What type of bonds can SDS break down? give examples
noncovalent bonds
Van der Waals
hydrogen bonds
ionic bonds
Why is SDS not sufficient in completely denaturing proteins?
because it only breaks down the noncovalent bonds present but there are also covalent bonds (disulfide bridges)
T or F: on its own, SDS is successful in denaturing quaternary proteins
false! it cannot break down the disulfide bridges
How can disulfide bridges be selectively disrupted without disrupting other covalent bonds?
the use of beta mercaptoethanol
What is beta ME? What is its purpose in SDS PAGE?
beta mercaptoethanol is a reducing agent that breaks down disulfide bridges between cysteine peptides and helps fully denature proteins
How is the gel plate orientated for polyacrylamide gels (including position of electrodes)? How are the samples loaded?
vertically with the negative cathode at the top and the positive anode at the bottom
Samples are loaded to separate wells at the top
Which direction do negatively charged samples run on an SDS PAGE?
from top to bottom (positive anode –> negative cathode)
How are the molecular masses of the samples determined?
by running a sample of known proteins with known molecular masses (a protein ladder/standard) to compare the unknowns to
What organism’s serum were proteins isolated from to use in the experiment?
bovine serum (Bos taurus)
Describe the structure of IgG
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
What other proteins are present in the bovine serum?
albumin pre-albumin acid glycoprotein haptoglobin macroglobulin
What were the 3 samples run on the SDS PAGE?
- protein ladder
- bovine serum treated with SDS only
- bovine serum treated with SDS + BME
