Labmath and Making Solutions

- The X Dilution Concentration

o Add X amount of concentrate to Y amounts of water to dilute stock
o To make 10 L of 1X TBE from 1 L 10X stock solution, add 1 L of stock and 9L of
water
- The Percent (%) Solution
o 1g in 100 ml = 1% weight/volume (w/v)
o 1 mL in 100 ml = 1% (volume/volume)
o An 80% sucrose solution means 80g of sucrose dissolved in a 100mL volume of
water
- Molarity Concentrations
o Molarity = moles/L
o Determine how many grams of a substance are needed to make a solution of a
desired molarity

g needed=(Formula weight g/mol)(Wolume desired L)(Molarity Desired mol/ L)
o Already have a solution but want to lower dilution
 C1V1 = C2V2
 C1 = the concentration of the starting stock solution
 C2 = the concentration of the new solution you are making
 V1 = the volume of the starting stock solution (C1) needed to make
the new solution
 V2 = the final volume of the new solution
Plasmid Purification
- Plasmids: extrachromosomal pieces of DNA that carry genetic information useful to the
bacterial cell
o Genes encoded often confer traits that enhance the ability of the bacteria to
survive under selective pressures
o Replication is associated with chromosomal replication
- Homologous expression: gene is expressed in its native organism
- Heterologous expression: gene is expressed in a non-native organism
- Plasmid Purification Steps
o Centrifuge culture to create a bacterial cell pellet
o Resuspend the pellet using Solution I (buffering agent: Tris-Cl, EDTA, dextrose)
 Tris-Cl has an alkaline pH8 to reduce electrostatic interactions between
DNA and DNA scaffolding proteins
 Dextrose prevents cell from lying from osmotic shock due to resuspension
 EDTA binds to any free metal ions in the solution to prevent activation of
enzymes that may degrade the DNA
o Add Solution II (NaOH, SDS), a lysis buffer to lyse the cell and release call cell
contents
  SDS is a detergent and helps break down the cell wall
 NaOH breaks the hydrogen bonds in DNA, denaturing it
 Plasmid (circular) DNA strands are still physically associated
 Chromosomal (linear) DNA strands are seperated
o Add Solution III (NaAc – sodium acetate)
 Neutralizes the solution
 Allows base pairing to reoccur
 Chromosomal DNA (linear) cannot base pair again efficiently due
to its linear nature and aggregates into a white precipitate.
 Plasmid DNA is able
to base-pair
efficiently.
o Centrifuge to separate precipitated
Chromosomal DNA from the
Plasmid DNA still in the solution.
o Ethanol is added to the solution to
precipitate the Plasmid DNA
o Plasmid DNA pellet is isolated using
centrifugation
- Agarose Gel Electrophoresis
o Used to separate fragments of DNA
o The higher the percentage of
agarose in gel, the less porous the
gel and the higher the resolving
power of the gel (the greater its
ability to separate DNA)
o DNA is negatively charged and when an electric field is applied DNA migrates to
the positively charged anode
o DNA migrates based on molecular shape and size
o The longer the fragment of DNA the slower it migrates through the gel; smaller
DNA fragments will migrate through the gel more rapidly than larger fragments
- Forms of DNA
o Linear
o Supercoiled
o Nicked
Restriction Digest
- Restriction enzymes: cleave double stranded DNA at specific recognition sites, thought
of as a bacterium’s defense system
o Recognition sites: ~4-8 base pairs in length, and are palindromes
- Sticky ends: leave single-stranded over hangs, ligated together more efficiently, direction
can be controlled (inserted in only one direction into plasmid)
- Blunt ends: leaves no overhang, ligated into plasmid in either direction

- Endonucleases: cleave within a DNA strand

- Exonucleases: cleave DNA progressively from ends
- Isoschizomers: restriction endonucleases that recognize the same sequence and cleave at
the same site
- Neoschizomers: restriction endonucleases that recognize the same sequence but cleave at
different sites
- A unit of restriction enzyme is the amount of enzyme needed to digest to completion 1µg
of λ DNA in a 50 µl reaction in 1 hour at 37°C
o The final volume of enzyme in any restriction digest should not be greater than
10% of the reaction volume because the glycerol can inhibit the function of the
restriction digest
o At least 1 µg of DNA should be used
- Bovine serum albumin stabilizes certain restriction enzymes in solution (not required)
- Star Activity: cleavage of sequences by a restriction endonuclease at sites slightly
different than the defined recognition sequence causes by non-standard digest conditions
o Causes:
 High glycerol concentration
 Too much enzyme for the amount of DNA present
 Low ionic strength (not enough salt)
 High pH
 Presence of DMSO, ethanol, and
other organic solvents
- High molecular weight species are at the top and
low molecular weight species are at the bottom
- Predicting DNA Segments Example
o Mixture of ScaI and BgII, cuts at 2177,
245, 1813
o Size 1: 2177-1813 = 364
o Size 2: 1813 – 245 = 1,568
o Size 3: 2686 – (364+1,568) = 754
- Estimating the quantity of DNA Present
o Use brightness to determine amount of
DNA present
 o Determine concentration of DNA in well
o Multiple concentration by amount of DNA in digest
Polyacrylamide Gel Electrophoresis of Serum Proteins
- Used when resolving proteins or DNA fragments of nearly similar size
- Higher resolving power than agarose gels, can resolve DNA molecules whose lengths
differ by as little as 0.1%
- Native Gel: protein migrate through gel in native confirmation, molecular weight
cannot be determined as size is not the only factor affecting migration rate (charge is as
well)
- SDS Gel: proteins are denatured to their primary linear form
o SDS denatures protein and coats it with an uniform negative charge
o Amount of SDS bound to the protein is proportional to the molecular weight of
the linear polypeptide and is independent of the actual amino acid sequence
(migrate only based on size)
o Glycosylated proteins are an exception – only apparent molecular weight can be
determined (sugar side-chains cause protein to migrate differently than would be
expected of actual weight)
- Two gels stacked edge to edge vertically
o Top gel is stacking gel, bottom gel is resolving gel
o As proteins migrate into resolving gel they roughly align by size
 Resolving gel is denser and provides greater separation of proteins
 Resolving power of the gel is determined by the ratio of bisacrylamide
(responsible for crosslinking strands of polyacrylamide) to acrylamide
 As ratio of bisacrylamide to acrylamide increases the pore size
decreases since the overall concentration of acrylamide the
solution increases
- Coomassie brilliant blue non-specifically stains proteins blue
- Sera Proteins
o Most abundant protein in the blood sera of mammals is albumin
- Western Blotting
o Method to identify a specific protein as some have very similar sizes and
molecular weights
o Primary antibody binds to the protein and a second antibody binds to the
primary to make the protein visible
o Steps
 Sample of interest is run on either a native or SDS polyacrylamide gel
 If protein must be folded to be recognized by antibody then native
system must be used
 After the gel has been run the protein is transferred to a PDVF or
nitrocellulose membrane through electrophoretic transfer
 Membrane is incubated in a prehybridization buffer for two hours up to
overnight
 Has blocking agents such as bovine serum albumin or dried milk is
used to bind on any available space on the membrane
 Prevents antibody from binding non-specifically to the membrane
 Primary antibody is added and allowed to hybridize to the target protein
on the membrane
 Membrane washed to remove excess of the primary antibody
 Membrane is incubated with a secondary antibody specific to the primary
antibody
 Secondary antibodies are typically labeled to allow an indirect way
of detecting the presence of the bound primary antibody
o Chemiluminescent, radiolabeled (most sensitive but least
safe), colorimetric (easily performed but less sensitive)
FOLDIT: Predicting Protein Folding and Structure
- Not possible to predict the native 3D structure
of a protein based on the primary, linear amino
acid sequence alone
- Humans are better at predicting protein structure
than computers
- Programs like FOLDIT bring us closer to being
able to make predictions and have proved useful
in solving structures
- Protein Structure
o Primary: linear structure of amino acids
o Secondary: folding of segments into
alpha-helix or beta-sheets
o Tertiary: folding of the alpha-helix and
beta-sheets
o Quaternary: interaction of multiple
polypeptide chains (subunits) into one functional protein
- Rules for Protein Folding
o Peptide bonds are planar and rigid (40% double bond character)
 Free rotation around the α-carbon
o No overlap between amino acid side chains
o Two side groups (R) with the same charge will be as far away as possible
- Folding Tendencies
o Amino acids with polar (charged) side chains tend to be on the surface of the
protein
o Non-polar (uncharged) amino acid side chains tend to be on the inside of the
protein
o Hydrogen bonds tend to form between adjacent peptide linkages, particularly
between a carbonyl oxygen and a hydrogen/nitrogen of the next peptide bond
o Sulfhydryl group of cysteine react together to form covalent disulfide linkages

α-Helix β-sheets
- Right-handed in nature: 3.6 - Hydrogen bonding between adjacent
residues/turn with a pitch of 5.4A peptide chains
- In a left-handed helix the amino - Almost fully extended but have a buckle
acid side chains contact the or pleat
polypeptide backbone too closely
Crystallography
- Difficult to predict tertiary/quaternary
structure of a protein using primary
amino acid sequence
- X-ray diffraction used to resolve
structures of larger proteins – requires
pure crystals of protein
o Analyze diffraction pattern of
crystals using Bragg’s Law to
determine position of atoms and
therefore structure of molecule
o Bragg’s Law: 2 d sin(θ) = n λ
o Minimum of 2A resolution needed to accurately analyze amino acid side chains
- Steps to Create Protein Model
o Obtain a suitable crystal of purified protein of interest
o Subject crystal to x-ray beam and capture diffraction pattern
o Transform diffraction data to yield model of protein
- Crystals used must be at least 0.2mm in size along an edge
- Factors that influence crystallization: salt conditions, pH, precipitating agents
o PEG serves as a crowing agent to aid assembly of protein into lattice
o NaCl helps keep protein soluble / increases supersaturation to precipitate protein
- Best conditions for crystallization are determined experimentally
- Facilitating Crystallization: Hanging Drop Method
o Concentrated protein suspended as hanging drop
over a well with a buffer of higher ionic strength
than that which the protein is suspended in
o Sealed environment allows for the solution in the
drop to equilibrate with the reservoir solution in
the well
o Reservoir solution has a higher ionic strength
pulling the solution from the drop to the reservoir
 o Decreased solution volume, increase concentration of protein which surpasses its
solubility and becomes supersaturated and precipitates
- Lysozyme: present in tears, saliva, and chicken egg whites
o Weakens bacterial cell walls, prevents bacterial infection
o Tetragonal crystals in NaCl/pH 4.8
- Salt crystals can sometimes form
o Perfect squared crystals
o Use dye to tell the difference, dye is absorbed by protein and excluded by salt
o PEG may considerably slow uptake of dye

Size Exclusion Chromatography

- Molecules are separated by their relative size and
molecular weight
- Move through resin based on Stokes radii – effective
radius of a molecule based on a perfect sphere
o Ex. two molecules of the same weight but
different shapes: sphere will move through
faster than rod
- Molecules are eluted form column from largest to
smallest
- Resin has porous beads in which smaller molecules
are trapped and larger molecules are excluded
- Size of pores depend on crosslinking/concentration of
agarose or polyacrylamide
- Polyacrylamide gels are more resistant to collapse than agarose
- Partition coefficient (Kav) is the measure of the degree of interaction of a molecule
with a column resin
(V e −V o) (V e−V o)
o K av = =
(V t −V o ) Vi
 Vo = void volume, volume
outside the beads
 Vi = space within the beads
(Vt – Vo)
 Ve = elution volume for the
sample
 Vt = the bed volume
- Vo determined using a molecule to interact
with resin at all – usually blue dextran
(large and completely excluded form
resin)
- Vt determined using a small tracking molecule – potassium chromate (small and flows
easily into pores – elutes last)
 - Create a graph of Kav values on the y-axis and the log of molecular weight on the x-
axis to determine the molecular weight of a species
Enzyme Kinetics
- Enzymes
o Exhibit substrate specificity
o Only catalyze reactions where substrate can bind to the enzyme active sit
o Return to original state once product is formed
o Helps bring together molecules into the same small space to increase chances of
the reaction occurring
- Peroxidase: enzyme that catalyzes a reaction in which hydrogen peroxide is converted to
water
o Substrate: hydrogen peroxide

 hydrogen peroxide is toxic to cells and is a byproduct of respiration

produced in the mitochondria
- Turnip Peroxidase: hydrogen peroxide is reduced by turnip peroxidase while guaiacol is
oxidized to the brown product tetraguaiacol
o Guaiacol is the reducing agent/hydrogen donor

o Use a colorimetric assay to record the change in color of the solution as

tetraguaiacol is produced to measure the rate of the reaction
 Measure at absorbance of 470 nm
- Thermodynamics: study of whether a reaction will occur, reaction will occur if it is
energetically favorable
- Kinetics: provides information on reaction mechanism and reaction pathway
o Factors that affect the kinetics of a reaction: enzyme concentration, substrate
concentration, temperature, pH and the presence of inhibitors
- Effect of pH
o Enzymes are proteins and each of its side chains has a specific pK
o Protonation of the side chains plays a role in the activity of the enzyme
o pH too low or too high can denature the enzyme by affecting side chain
protonation and render it inactive
- Michaelis-Menten Curve
o Describes the rate of the reaction as a function of substrate concentration

V [ S ] max
vo =
K M +[ S ]

o Vmax is the maximum velocity of a reaction where the substrate concentration is

saturated (all active sites on enzyme are filled with substrate and the reaction
cannot occur any faster)
o KM is the Michaelis constant
o Km is the substrate concentration when the velocity of the reaction is half of the
Vmax
- Lineweaver-Burk Plot: linearization of Michaelis-Menten

1 Km 1 1
=( ) +
v o V max [ S ] V max

o Slope = Km/Vmax
o X-intercept = -1/Km
 o Y-intercept = 1/Vmax
o The larger the substrate the concentration the closer to the y-axis 1/[S] values
- Inhibitors of Enzyme Activity
o Competitive Inhibitor: directly competes with the substrate for the active site of
the enzyme, enzyme binds the inhibitor but cannot function in the same way as it
would with the substrate

 As the concentration of the inhibitor increases the velocity of the reaction

decreases but the overall Vmax stays the same
 Lines will intersect on the y-axis in a Lineweaver-Burk plot
 X-intercept = -1/αKm where α = (1+[I]/KI)
o [I] is the inhibitor concentration
o KI is the dissociation constant for inhibitor binding
o KI = [E][I]/[EI]
o Uncompetitive Inhibitor: binds to the enzyme-substrate complex (ES) but not to
the free enzyme, interferes with catalytic function of enzyme likely by altering the
shape of protein and active site but doesn’t actually bind to the active site
  Lines on Lineweaver-Burk plot are parallel to each other
 KI’ is the dissociation constant
 α' = (1+[I]/KI’)
o Mixed Inhibition: enzyme bins to both the enzyme and the enzyme-substrate
(ES) complex
  On a Lineweaver-Burk plot the lines intersect to the left of the y-axis
o Noncompetitive Inhibition: inhibitor binds the enzyme and the enzyme-substrate
(ES) complex with equal affinity
 Binding of the inhibitor to the enzyme does not change the enzymes
affinity for the substrate and the binding of the substrate does not change
the binding affinity of the inhibitor
 Lines will intersect to the left of the y-axis and on the x-axis itself
Hill Reaction
- Photosynthesis: synthesis using light; conversion of light energy to chemical energy by
photosynthetic pigments using water and CO2 to produce carbohydrates

6CO2 + 6H2O → C6H12O6 + 6O2

- Chloroplasts
o Located in mesophyll (between upper and lower epidermis)
o Thylakoid reactions
 Occur in thylakoid of chloroplast
 Produce ATP and NADPH
 Referred to as light reactions
o Carbon fixation reactions (Calvin Cycle)
 Occur in the chloroplast stroma
 Synthesis of sugar
 Don’t require light “dark reactions”
- Photon: a discrete particle of light energy
o Electrons for ETC do not come from light
 o Quantum: the amount of energy (E) in a photon
 E = hhv
o Chlorophyll → blue and red light
 Absorbance drives ETC
 Light excites electrons
- Chlorophyll: key feature is MG in the middle, tail ties molecule to thylakoid membrane
o Chlorophyll a – methyl group
o Chlorophyll b – aldehyde
- Carotenoids
o Found in all photosynthetic organisms
o Located in thylakoid membrane
o Light absorbed is transferred to chlorophyll
o Protection from light damage (accessory protein)
- Antenna Complex: a group of pigment molecules that cooperate to absorb light energy
and transfer it to a reaction center complex. Chlorophylls and carotenoids are a part of the
complex
- Redox reactions: electrons are removed from one species (oxidizing it) and added to
another species (reducing it)
- Hill Reaction: water serves as the source of electrons for photosynthesis, NADP+ serves
as the final electron acceptor by reduction to NADPH

- Two Photosystems:
o Photosystem I: system of photoreactions that absorbs maximally far-red light
(>680 nm), oxidizes plastocyanin and reduces ferredoxin
o Photosystem II: system of photoreactions that absorb maximally red light (680
nm), oxidizes water and reduces plastoquinone, operates very poorly under red
light
- Electrons are moved in the ETC using carriers
o Plastoquinone: diffusible electron carrier located in thylakoid membrane
o Plastocyanin: diffusible electron carrier located in thylakoid lumen
o Cytochrome b6f: evenly distributed through stacked grana and stoma lamellae
- Steps:
o A photon of light hits a chlorophyll molecule surrounding the Photosystem II
complex creating resonance energy that is transferred between surrounding
chlorophylls to the reaction center of Photosystem II
o When the energy reaches the reaction center, an electron is released. One photon
is required for each electron.
o Two excited electrons are transferred to plastoquinone QB (first mobile carrier)
 Plastoquinone QB also picks up two protons
o To replace the two electrons lost at the reaction center of Photosystem II, water is
split to produce hydrogen, oxygen, and two electrons
o Plastoquinone QB transfers the two electrons to the Cytochrome b6f complex,
and the two protons it picked up are released into the lumen
 Cytochrome b6f also transfers two protons into the lumen
o The two electrons are transferred to plastocyanin
o The two electrons are transferred from plastocyanin to the Photosystem I
complex
o Photons energize each electron in the reaction center of Photosystem I and propel
their transfer to ferredoxin
o Ferredoxin then transfers the electrons to ferredoxin NADP reductase (FNR)
o After two electrons are transferred to FNR, NADP+ is reduced to NADPH
o The gradient created by the electron transport chain is utilized by ATP synthase
to produce ATP from ADP and Pi