MCAT Lab Techniques
MCAT Lab Techniques
MCAT Lab Techniques
IWantAHighMCATScore’s
Guide to MCAT Lab Techniques
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Contents
Gel Electrophoresis ........................................................................................................................................ 3
Native-PAGE .............................................................................................................................................. 3
SDS-PAGE .................................................................................................................................................. 3
Reducing SDS-PAGE ................................................................................................................................... 4
Isoelectric Focusing .................................................................................................................................... 4
Western Blotting (Protein) ............................................................................................................................. 4
Southern (DNA) and Northern (RNA) Blotting .................................................................................................. 5
DNA Sequencing (Sanger Dideoxynucleotide Sequencing) ................................................................................ 6
Chromatography............................................................................................................................................ 7
Liquid Chromatography .............................................................................................................................. 7
High-Performance Liquid Chromatography (HPLC) ....................................................................................... 7
Gas Chromatography ................................................................................................................................. 7
Gel-Filtration (Size Exclusion) Chromatography............................................................................................ 7
Ion-Exchange Chromatography ................................................................................................................... 8
Affinity Chromatography ............................................................................................................................ 8
Thin-Layer Chromatography ....................................................................................................................... 8
Distillation .................................................................................................................................................... 9
Simple Distillation ...................................................................................................................................... 9
Fractional Distillation ................................................................................................................................. 9
Vacuum Distillation .................................................................................................................................... 9
Polymerase Chain Reaction ............................................................................................................................ 9
Spectroscopy ............................................................................................................................................... 10
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H-NMR Spectroscopy .............................................................................................................................. 10
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C-NMR spectroscopy ............................................................................................................................. 11
IR Spectroscopy ....................................................................................................................................... 11
UV-Vis Spectroscopy ................................................................................................................................ 11
Autoradiography ......................................................................................................................................... 12
X-Ray Crystallography .................................................................................................................................. 12
Immunoprecipitation ................................................................................................................................... 12
Radioimmunoassay ...................................................................................................................................... 12
Mass Spectrometry ...................................................................................................................................... 13
Enzyme-Linked Immunosorbent Assay (ELISA) ............................................................................................... 14
Indirect ELISA .......................................................................................................................................... 14
Sandwich ELISA........................................................................................................................................ 14
Edman Degradation ..................................................................................................................................... 15
Gram Staining .............................................................................................................................................. 15
Restriction Fragment Length Polymorphism (RFLP) ........................................................................................ 16
Salting Out and Dialysis ................................................................................................................................ 16
Reducing Sugar Tests ................................................................................................................................... 17
Tollen’s Test ............................................................................................................................................ 17
Benedict’s Test ........................................................................................................................................ 17
Fehling’s Test........................................................................................................................................... 17
cDNA Libraries ............................................................................................................................................. 18
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Gel Electrophoresis
Macromolecules (proteins, DNA, or RNA) of interest are placed in the lanes of a gel. For proteins and
small molecules of DNA and RNA, the gel will be polyacrylamide. For larger molecules of DNA (> 500 bp),
the gel will be agarose. An electrical charge is placed across the gel. At the bottom is the positively
charged anode and at the top is the negatively charged cathode. Keep in mind, since a voltage source is
applied to gel electrophoresis, it follows the same principles as an electrolytic cell. Negatively charged
molecules will travel toward the anode. Because of the resistance of the gel, larger molecules will have a
harder time moving and thus, the molecules will be separated by size with the smallest molecules
toward the bottom. The gel can then be stained for visualization, typically using Coomassie Blue dye. A
lane will be loaded with a collection of molecules of a known size, called a ladder, which can used to
determine the size of the molecules being ran. There are several different applications of gel
electrophoresis:
Native-PAGE
Native-PAGE is a polyacrylamide gel electrophoresis method for proteins that occurs under non-
denaturing conditions. This method will
separate proteins by size while retaining
their structure.
SDS-PAGE
SDS-PAGE is a polyacrylamide gel
electrophoresis method for proteins that
occurs under denaturing conditions to separate proteins by mass. Negatively-charged sodium dodecyl
sulfate (SDS) is added to the solution of proteins, denatures the proteins, and binds one SDS for every
two amino acids, giving all proteins the same charge-to-mass ratio. Since all proteins have the same
charge-to-mass ratio, they are separated solely on mass, with the smallest proteins found toward the
bottom of the gel. SDS will only interrupt non-covalent bonds, so if disulfide bridges are present in the
protein, they will not be broken. This is useful when analyzing proteins with multiple subunits.
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Reducing SDS-PAGE
Reducing SDS-PAGE is exactly like SDS-PAGE, but with the addition of a reducing agent, like β-
mercaptoethanol, which will reduce disulfide bridges and result in a completely denatured protein.
Isoelectric Focusing
Isoelectric focusing is a gel
electrophoresis method that separates
proteins on the basis of their relative
contents of acidic and basic residues. A
polyacrylamide gel with a pH gradient
(low pH on one side, high pH on the
other) is used. When proteins migrate
through the pH gradient gel, they will
travel toward the anode until the area
of the gel with the pH that matches their isoelectric point (pI). When a protein is at its pI, it has a net
charge of zero and will not be attracted to the positively charged anode so it will not move.
Step 1: Proteins from a sample are loaded into an SDS-PAGE gel and separated based on size
Step 2: Proteins from the gel are transferred to a polymer sheet and exposed to a radiolabeled antibody
(sometimes using two antibodies; one specific to the protein of interest and another radiolabeled
antibody that binds to the first antibody) that is specific to our protein of interest
Step 3: The polymer sheet is viewed used autoradiography. The protein of interest that is bound to the
radiolabeled antibody will be visible.
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Purpose: Detection of a specific DNA (Southern blot) or RNA (Northern blot) sequence in a sample
Step 1: The DNA strand of interest is exposed to restriction enzymes that cut the DNA strand into
smaller fragments
Step 2: The newly cleaved strands of DNA are denatured using a solution of NaOH to create ssDNA
strands
Step 3: The single stranded cleaved strands of DNA undergo gel electrophoresis, separating them by
size. Smaller fragments will be found at the bottom of the gel. Polyacrylamide is used if the stands are
less than 500 base pairs. Agarose is used if the strands are over 500 base pairs.
Step 4: The DNA from the gel is transferred to a sheet of nitrocellulose paper and then exposed to a 32P
radiolabeled DNA probe that is complementary to our DNA of interest.
Step 5: The nitrocellulose paper is then viewed using autoradiography to identify the strand of interest.
NOTE: These methods are nearly identical for Southern and Northern blotting. The steps listed above
are for Southern blotting, however, the only difference is that Northern blotting uses RNA, so steps 1
and 2 are not done.
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Modified nucleotides, known as “dideoxynucleotides” (ddNTPs), are used in this method. ddNTPs are
missing the OH group on the 3’ carbon, thus they are unable to create a new 5’→3’ phosphodiester
bond. This allows us to control the termination of replication.
Step 1: The DNA strand of interest is denatured using an NaOH solution to create a ssDNA strand that
we can use for replication
For each respective nucleotide, the insertion of a ddNTP will terminate replication and create various
strands of different length that correspond to that specific nucleotide. Therefore, the gel can be read
from bottom-to-top to determine the nucleotide sequence. The smaller the fragment, the further it
travels in the gel.
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Chromatography
There are several different types of chromatography that can be used for separating or analyzing a
mixture of two or more molecules based on their properties. Traditionally, there are two components to
chromatography; a stationary phase, which is typically polar, and a mobile phase, which is typically non-
polar. Polar molecules are separated from a mixture by staying with the stationary phase, while non-
polar molecules stay with the mobile phase. However, if reverse-phase is specified, then the properties
of the two phases are switched. This also changes based on the type of
chromatography used, as some methods use ligands or gel beads.
Liquid Chromatography
In liquid chromatography, silica is traditionally used as the stationary
phase while toluene or another non-polar liquid is used as the mobile
phase.
Gas Chromatography
Gas chromatography (also known as gas-liquid chromatography) is used to separate and analyze
molecules that can be vaporized. The mobile phase is an inert or unreactive gas, such as helium or
nitrogen, while the stationary phase is a thin layer of liquid or polymer that surrounds the walls of a
tube. The stationary phase allows more polar molecules to elute slower, giving them a higher retention
time.
Ion-Exchange Chromatography
Ion-exchange chromatography will separate proteins by their net charge. The
column is filled with charged beads, either positive or negative. In cation-
exchange, negatively-charged beads are used which attract positively charged
proteins and negatively-charged proteins will elute first. In anion-exchange,
positively-charged beads are used which attract negatively-charged proteins
and positively-charged proteins will elute first.
Affinity Chromatography
Affinity chromatography will separate proteins based on their affinity
for a specific ligand. Beads that are bound to a specific ligand will be
used and proteins with a high affinity for that ligand will bind to the
beads, allowing proteins with a low affinity to elute first. The high
affinity proteins are then eluted by increasing the concentration of
the free ligand in the column, which competes for the active site of
the bound proteins.
Thin-Layer Chromatography
Thin-layer chromatography consists of a
small sheet of medium that is coated in an
adsorbent material, such as silica gel. The
polar silica is the stationary phase. The
molecules of interest are added to the
bottom of the sheet and the sheet is
placed in a non-polar liquid, such as
heptane, until it reaches the origin. The
mobile phase then travels up the plate
using capillary action, allowing the
molecules to move with it if they are
relatively non-polar. The spots are then
visualized using UV light.
The relative distances traveled between the molecules is represented by the Rf value, which is measured
as the ratio of the distance the molecule traveled from the origin to the distance the solvent front
traveled from the origin.
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Distillation
Simple Distillation
Simple distillation is used to separate two molecules from a solution when their boiling points differ by
25o C or greater.
Fractional Distillation
Fractional distillation is used to separate two molecules from a solution when their boiling points differ
by less than 25o C.
Vacuum Distillation
Vacuum distillation is used to separate two molecules from a solution when their boiling points are high
and risk changing chemically.
Spectroscopy
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H-NMR Spectroscopy
The application of nuclear magnetic
resonance regarding the 1H isotope
within the molecules of a substance.
Integration: The integration of the peak determines the number of equivalent hydrogens a signal
represents.
Neighbors: The number of peaks determines the number of neighboring hydrogens that are ≤ 3 bonds
away. The number of peaks equals the number of neighbors + 1.
- Singlet → No neighboring hydrogens
- Doublet → One neighboring hydrogen
- Triplet → Two neighboring hydrogens
- Quartet → Three neighboring hydrogens
- Quintet → Four neighboring hydrogens
- Sextet → Five neighboring hydrogens
- Septet → Six neighboring hydrogens
- Mul plet → Seven or more neighboring hydrogens
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C-NMR spectroscopy
The application of nuclear magnetic resonance regarding the 13C isotope within the molecules of a
substance
Chemical shift: The chemical shift on the x-axis (δppm) represents the amount of deshielding of
electrons that is caused by an adjacent heteroatom or pi bond.
- 0 - 70 ppm → Alkane region
- 90 - 120 ppm → Alkene region
- 110 - 160 ppm → Aroma c region
- 160 - 200 ppm → Carbonyl region
IR Spectroscopy
IR spectroscopy is a method that
is used to identify certain
functional groups within a
molecule. Only molecules that
have a dipole moment will show
absorbance. The x-axis is reported
in wavenumbers (reciprocal
centimeters) and the y-axis is
reported in percent absorbance.
Important regions:
- 1700 - 1750 → Carbonyls (sharp peak)
- 1720 - 1740 → Aldehydes
- 1700 - 1725 → Ketones
- 1735 - 1750 → Esters
- 1700 - 1725 → Carboxylic acids
- 3200 - 3600 → OH groups (broad peak)
- 3300 - 3400 → Amines
- The number of peaks are relative to the number of hydrogens on the amine (e.g., 1o
amines will have two peaks, 2o amines
will have one peak)
UV-Vis Spectroscopy
As the number of conjugated pi bonds increase, the
energy gap between the highest occupied molecular
orbital (HOMO) and lowest unoccupied molecular
orbital (LUMO) decreases, which means that light of a
lower energy is absorbed. This results in light with a
longer wavelength to be emitted (as seen on the right).
If a molecule absorbs green light, we see it as red.
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Autoradiography
In the scope of the MCAT, autoradiography is used primarily as an imaging technique to identify
radiolabeled atoms that are in a molecule, often as part of a Southern, Northern, or Western blot. The
radiolabeled substance is placed in contact with a photographic emulsion containing silver halide
crystals. The radiation of the radiolabeled substance converts the silver halide crystals into metallic
silver, producing an image.
X-Ray Crystallography
In x-ray crystallography, a crystalline molecule causes a beam of incident x-rays to diffract into many
specific directions. A three-dimensional image can be constructed by measuring the different angles and
intensities of the diffracted rays.
Immunoprecipitation
Radioimmunoassay
Similar to ELISA, the wells of a plate are coated with the primary antibody that is specific to our protein
of interest. Next, the radiolabeled protein, typically containing a tyrosine residue labeled with 125I, is
added to the wells and binds to the primary antibody. The concentration of that radiolabeled protein is
determined by counting the gamma emission. Next, a sample of the protein of interest in an unknown
concentration is added to the wells, where it competes for the active site on the primary antibody,
displacing an amount of the radiolabeled protein. The concentration of the radiolabeled protein is
determined again by counting the gamma emission. The difference in concentrations provides the
concentration of the protein of interest.
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Mass Spectrometry
Base peak: The tallest peak. This does not always represent the actual intact molecule, as it may
sometimes be a fragment of the molecule that is found in higher abundance.
Molecular ion peak (M): The peak that represents the molecule. The m/z value of this peak represents
the molecular weight of the molecule.
M+1 peak: The relative abundance of 13C in the molecule. Found in a relative abundance of 1.1%. So if
there is a M+1 with a m/z value of 4.4, it means that there are 4 carbons present (4.4/1.1 = 4).
M+2 peak: The relative abundance of either 37Cl or 81Br in the molecule. 37Cl will be found in a 3:1 ratio
relative to the M peak (e.g., if the M peak has a relative abundance of 90%, if the M+2 peak is at 30%, it
means there is chlorine present in the molecule). 81Br will be found in a 1:1 ratio relative to the M peak
(e.g., if the M peak has a relative abundance of 90%, if the M+2 peak is also at 90%, it means there is
bromine present in the molecule).
Fragments: There are three types of fragmentation that can occur, which can help identify the structure
of the molecule. 1) Alkane fragmentation, 2) alcohol dehydration, and 3) alpha cleavage. These are most
likely beyond the scope of the MCAT.
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Indirect ELISA
The molecule of interest (in this case, considered an antigen) is coated to the wells of a plate. A primary
antibody that is specific for that antigen is added and washed to remove any unbound antibodies. Next,
the secondary antibody is added. The secondary antibody is linked to an enzyme, often horseradish
peroxidase (HRP), that will be converted into a fluorophore when reacted with an oxidizing agent, such
as hydrogen peroxide. This will cause a change in color, which can be detected using
photospectrometry. The absorbance of the well is compared against a serial-diluted standard of known
concentration, and based on a determined curve using the standards, the concentration of the molecule
in the well can be determined.
Sandwich ELISA
The primary antibody that is specific to our molecule of interest is coated to the wells of a plate. Next,
the molecule of interest is added to the wells and then washed to remove any unbound molecules.
Next, the secondary antibody is added. The secondary antibody is linked to an enzyme, often
horseradish peroxidase (HRP), that will be converted into a fluorophore when reacted with an oxidizing
agent, such as hydrogen peroxide. This will cause a change in color, which can be detected using
photospectrometry. The absorbance of the well is compared against a serial-diluted standard of known
concentration, and based on a determined curve using the standards, the concentration of the molecule
in the well can be determined.
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Edman Degradation
Gram Staining
Purpose: Used to differentiate bacteria into two groups, gram-positive or gram-negative, based on the
content of their cell wall
The sample of bacteria is heat-fixed to a slide and crystal violet dye is added. Iodide is added to the
bacteria, which binds to the crystal violet dye and traps it in the bacterial cell. The bacteria is then
washed with alcohol to remove any dye that was not taken up by the bacteria. Safranin is added as a
counterstain, so that the bacteria that did not stain with crystal violet can be visualized.
Gram-positive bacteria: Appears purple on the slide. Contains a thick peptidoglycan layer that takes up
the stain.
Gram-negative bacteria: Appears pink on the slide. Contains a thin peptidoglycan layer sandwiched
between two lipid bilayers that take up the safranin stain.
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Tollen’s Test
Tollen’s reagent tests for the presence of an aldehyde and can distinguish between aldoses and ketoses.
Ketones do not react unless they are α-hydroxy-ketones. A positive Tollen’s test is characterized by the
precipitation of elemental silver.
Benedict’s Test
Benedict’s reagent tests for the presence of an aldehyde. Ketones do not react unless they are α-
hydroxy-ketones. A positive Benedict’s test is characterized by a change in color from clear blue to brick-
red with the formation of a precipitate.
Benedict’s reagent consists of a mixture of sodium carbonate, sodium citrate, and copper (II) sulfate
pentahydrate.
Fehling’s Test
Fehling’s solution tests for the presence of an aldehyde. Ketones do not react unless they are α-hydroxy-
ketones. A positive Fehling’s test is characterized by a change in color from clear blue to brick-red with
the formation of a precipitate.
Fehling’s solution consists of two parts; Fehling’s A and Fehling’s B. Fehling’s A consists of aqueous
copper (II) sulfate. Fehling’s B consists of potassium sodium tartrate and sodium hydroxide.
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cDNA Libraries
Purpose: Used to create and house complimentary DNA (cDNA) strands that can be expressed in
bacterial vectors
In order to synthesize proteins for things like medication, such as insulin, we must clone the DNA
sequence for that protein in a bacterial vector. However, eukaryotes have the ability to undergo post-
transcriptional modification of hnRNA to create mRNA while prokaryotes do not. Therefore, we can not
insert eukaryotic DNA directly into a bacterial vector since it still contains introns. Instead, we must
synthesize a strand of cDNA from the protein of interest’s respective mRNA.
The mRNA transcript of the protein of interest is isolated and a complementary DNA primer containing
many thymine repeats and a free 3’-OH group is added, which anneals to the poly-A tail of the mRNA.
Next, the mRNA is exposed to reverse transcriptase and an excess of the four dNTPs so a
complementary cDNA strand can be synthesized, creating a cDNA-mRNA hybrid. The hybrid strand is
hydrolyzed using an alkaline solution. A primer is added to the new single-stranded cDNA and the
complementary strand is synthesized by DNA polymerase. The new double-stranded cDNA can be
inserted into a bacterial plasmid vector by using restriction enzymes and the bacteria will synthesize the
protein of interest.