Biochemistry Lab Practical
Micropipette range
*check pipette top as well as matching color with tips* -remember to lock and unlock -1st stop to draw water -second stop to release
uncompetitive inhibition (lineweaver burk plot)
- Both Km and Vmax affected - "Apparent" Vmax less than true Vmax - "Apparent" Km less than true Km
Fold Dilutions
-2 fold dilution = 1/2 stock strength = ½ stock +½ diluent -4 fold dilution = 1/4 stock strength = ¼ stock + ¾ diluent
WHY is BME important?
-BME breaks the disulfide bonds, removing tertiary and quaternary structure
Why is EDTA used as the final step in the Western Blot procedure?
-EDTA is a chelating agent and will sequester metal ions and thus stop the alkaline phosphatase reaction
For the kinetics lab, you will be determining the kinetic parameters for alkaline phosphatase from both Lineweaver-Burk and Michaelis-Menten plots. Which of the following TWO statements below are CORRECT?
-Excel must be used to create all graphs. -The Michaelis-Menten plot will be generated using non-linear regression analysis using Excel. This plot is preferred over the Lineweaver-Burk plot to determine kinetic parameters because the Lineweaver-Burk plot introduces error when the data is linearized.
antibody-antigen interaction
-Fab domain: binds to specific antigen -Fc domain: binds to secondary antibody domain
Lactate dehydrogenase is an enzyme which catalyzes an irreversible oxidation-reduction converting pyruvate to lactate.
-False
Primary antibodies are not protein specific. The same primary antibody will be used to detect cytochrome c, ovalbumin, and beta-galactosidase.
-False
ion exchange chromatography
-IEC retains molecules based on ionic interactions -stationary phase proteins ionic functional groups which attracts molecules of opposite charge --further divided into: -cation exchange chromatography -anion exchange chromatography
Molarity
-Number of moles of solute in exactly one liter of a solution. -Units: moles/L
For the Separation of Protein series of labs, what two methods will be used to determine the concentration of ovalbumin and cytochrome c in your peak fractions (select two answers!)?
-Pierce 660nm protein assay -Absorbance at 280nm will be measured and used to calculate the protein concentration
Which of the following statements below are correct (select all statements that are correct!).
-SDS coats protein backbone with a negative charge -SDS linearizes the protein -SDS removes secondary structure from proteins
why is SDS important for protein preparation?
-SDS coats protein backbone with a negative charge and linearizes the protein, removing secondary structure -separation of denatured protein is only based on size
Large Transfers
-Serological pipettes -Graduated cylinders
Meniscus can be seen in...
-Serological pipettes -Graduated cylinders
Small transfers
-Transfer pipettes -Pasteur pipettes -Micropipettes Accurate & Precise
Three Concluding Statements are required for the Antioxidant Lab Report.
-True
absorbance
-UNITLESS metric of how much light is absorbed -absorbance can be measured at any wavelength, but will be highest at the OPTIMAL WAVELENGTH, which will give you the best resolution on a TLC sheet if the solvent front travels further
enzyme catalysis
-a catalyst is a substance that increases the rate of a rxn -enzymes do this by reducing the energy of activation of a particular reaction, and there by accelerating the formation of products from reactants and vice-verse
antioxidants
-a substance that delays or prevents oxidation of a substrate
extinction coefficient
-absorbance depends on E -E is unique to each substance -E depends on the intrinisc properties of the substance being measured -Example: Peptides; E is largely dependent on aromatic residues such as tryptophan and tyrosine
Western blotting
-allows you to identify proteins of interest with specific antibodies
protein gel electrophoresis methods
-always work with BME in hood -vortex sample -centrifuge sample (balance them with equal volume) -heat sample at 90 degrees C -rinse wells with buffer -load wells (based on directions follow carefully) -run gel -place gel fragments in labeled coomasise stain and transfer buffer containers -transfer apparatus -prepare sandwich -labeling transfer apparatus -remove blot from transfer apparatus
what is a free radical?
-an atom containing at least one unpaired electron in its outermost shell -reactive oxygen species (ROS): free radicals derived from molecular oxygen
cellular defense mechanisms
-antioxidant scavenging enzymes: --superoxide dismutase --catalase --glutathione peroxidase -repair processes -degradation of damaged proteins and apoptosis
size exclusion chromatography principle
-beads in column have tiny pores -the mixture of molecules is added to the column -large molecules move through the column quickly traveling around the beads -smaller molecules move through the pores of the beads and take longer to pass through the column
Pipetting Potential Errors
-bubbles in tip -drawing up at an angle -2 stops before drawing up -submerging tip before depressing plunger -not using 2nd stop for dispensing -tipped filled pipette
Alkaline phosphatase
-catalyzes the hydrolysis of esters of phosphoric acids -In bacteria, it is involved in the phosphate starvation stress response pathway. In eukaryotes...not really sure. -
lactate dehydrogenase (LDH)
-converts pyruvate to lactate-reversible, oxidation-reduction reaction -conversion to lactate favored in anaerobic conditions -co-enzyme NAD+ formed in forward reaction, required for glycolysis -reverse reaction occurs in the liver, NAD+ is converted to NADH
How do antioxidants prevent oxidation?
-directly inhibits oxidation reaction by becoming oxidized themselves -trap unstable free radical intermediates
wavelength
-distance of a repeating part of a wave (for light, measured in nm) -in photometry, we look for an OPTIMAL WAVELENGTH to absorb light (peak absorbance)
pierce 660nm assay: protein quantification
-dye metal complex based colormetric assay --concentration of protein related to color change monitored by spectrophotometry -used for fast and accurate and more reliable protein concentration measurements -protein concentration range of 25-20000 microgram/mL -presence of detergents/reducing agents does not interfere with the assay -low volume of sample
E. coli lysate to demonstrate western blot advantages
-e. coli cells make an excess amount of the enzyme that breaks down the sugar b-galactose -E. coli are lysed to spill the contents of the cells -when run on a gel, you will see the total proteins expressed by the cell at the time of lysis -over-expressed proteins, will appear darker than other bands on the gel -western blotting allows for you to determine if your protein of interest in your lysate, when it is difficult to determine from the gel
classification of antioxidants
-enzymatic (Antioxidant scavening enzymes) --superoxide dismutase -catalase -glutathione peroxidase -glutathione reductase non-enzymatic (free-radical scavengers): endogenous: uric acid, melatonin exogenous: vitamin E, vitamin C, and carotenoids
absorption spectrum
-every substance has a unique absorption spectrum -lamda max: wavelength at which max amount of light is absorbed by solution
LDH expression
-gene expressing LDH from lactobacilius acidophilus cloned in E. coli - overexpressed LDH -LDH protein tagged with His residues that will interact with Ni-NTA column
elution of bound LDH
-high concentratioons of imidazole in the buffers competes with His residues to bind nickel ions in stationary phase -imidazole replaces the bound protein by binding to the column itself
different types of chromatography
-ion exchange chromatography -gel filtration chromatography -affinity chromatography
IEC common errors
-loading the sample too quickly -getting a bubble in the column -disrupting the column bed -letting column dry -inaccurate fraction monitoring -loading buffers in wrong order -disposing of fractions before spotting
GFC matrix selection
-matrices have different fractionation ranges, or the ranges of molecular weights the column can accommodate -should include the protein of interest
spectrophotometer
-measures the fraction of light transmitted through a solution as a function of wavelength -some uses: -determine protein concentration -assay enzymatic activity -characterize chemical properties
SDS (sodium dodecyl sulfate gel electrophoresis)
-method separating denatured proteins based on their size -methods: --determine protein size --identify protein --quantify protein --starting point for western blots
cation exchange chromatography
-negatively charged stationary phase -binds positively charged molecules (cations) -repels and elutes, negatively charged molecules -removal of bound ions by changing salt concentration or pH
Errors in sep of proteins part 2
-not denaturing sample before loading: BME, sample buffer, or heat -not loading sample to bottom of well -piercing gel with pipette tip -loading sample outside well -loading sample across multiple wells -forgetting PVDF membrane in sandwich
separation of proteins
-ovalbumin (43 kDa) -cytochrome c (12 kDa)
ROS can be damaged to the cell
-oxidative stress: when the amount of ROS produced exceeds -the rate of removal by cellular defense mechanisms -leads to damaged lipids, DNA/nucleic acids, and proteins -linked to Alzheimer's disease, cancer, heart disease, Parkinson disease and others
p-nitrophenol
-p-nitrophenol: colorless at low pH; unionized -phenolate ion yellow color at high pH; ionized
What is pH?
-pH = -log {H+] -pOH = -log [-OH] [H3O+] = [H+] acid = 1x10^-7M [OH-] = base = 1 x 10^-7 M kw= [H+] [-OH]= 1x10^-14 M pH + pOH = 14
pI or Isoelectric point
-pH at which the net charge of the amino acid is zero (neutral charge). -amino acid is entirely in zwitterion form -if the amino acid DOES not have an ionizable side chain, it will be mostly in zwitterion form at neutral pH -To calculate pl, average the two pKa values where the amino acid comes from + -> zwitterion and from zwitterion -> -
dietary antioxidants
-plant derived polyphenols --found in fruits, nuts, vegetables, wine, tea, and coffee --contain readily oxidizable (-OH) substitutions which can act as antioxidants -vitamin E, vitamin C, carotenoid, and the mineral selenium
anion exchange chromatography
-positively charged stationary phase -binds negatively charged molecules (Anions) -repels, and elutes, positively charged molecules -removal of bound ions by changing salt concentration or pH
thin layer chromatography
-prepare TLC plate -label Xs -spot with amino acids and dry -Do not touch plate with pipette tip -repeat spotting and dry -place plate in tank -after 2 hours, remove plate from tank -mark migration front and re-label plate TLC: potential errors -use care to not scratch plate
How to use a pH meter
-press standby to turn on meter -remove probe form storage solution and rinse -place probe in standard solution -wait for meter to stabilize -remove probe and rinse -repeat for all standard solutions -"good electrode" = ready to measure sample -rinse probe and return to storage solution ERRORS: -DO NOT PRESS STD when measuring samples
Free radicals derived from molecular oxygen are referred to as__________________________
-reactive oxygen species
When performing the Antioxidant lab, you will test the ability of substances to inhibit the______________ of nitro blue tetrazolium (NBT)?
-reduction
Aliquot
-refers to a small sample taken from a larger volume -often aliquots are taken at each step of a protocol (such as purification of protein) and used to analyze the success of the protocol
precision
-reproducibility -the degree by which repeated measurements yield similar results
absorbance at 280 nm
-required additional info -affected by sample impurities
Proper Use of Fraction Collector and Pump
-rotate drop arm to position -remove carousel -load carousel with fraction collection tubes -replace loaded carousel -engage carousel engages gear drive -rotate drop arm to collecting position -fraction pump -flush pump tubing -connect pump tubing to collector tubing -flush collector tubing -press run simultaneously -press run on the pump and collector -measure samples as they clear the drop arm -measure sample absorbance at multiple wavelengths (measure sample each time)
preparing protein samples for SDS page
-sample buffer: tris Cl buffer -> allows to keep pH 6.8 -SDS -beta mercaptoethanol (BME) -> breaks disulfide bonds -glycerol -> increases density of sample -bromophenol blue - track samples --heat then centrifuge
kinetics: alkaline phosphatase
-substrate: p-nitrophenyl phosphate -product: phenolate ion, inorganic phosphate -enzyme: alkaline phosphatase -inhibitor: orthophosphate
structure of LDH
-tetramer = 4 subunits -35, 531 Da
protein quantification
-the assay uses polyhydroxylbenzenesulfonephthalein type dye and a transition metal complex based total determination method -the dye-metal complex has a reddish brown color that changes to green on binding to protein -there is a shift in the absorption maximum of the dye-metal complex from 450-660 nm
separating an amino acid mixture
-the functional group of each amino aid can vary in charge, dependent on the surrounding environment -a change in pH can alter the charge of an amino acid's functional groups -by increasing pH, you can selectively elute each amino acid via IEC
After proteins are transferred to the membrane, the membrane will be incubated in a blocking solution containing 10% milk. What does milk contain that is able to bind sites on the membrane that are not occupied by protein transferred from the SDS-PAGE gel?
-the protein casein
affinity chromatography
-utilizes specific interactions between molecules --receptor ligand --antigen-antibody -interactions could be varying in nature ionic, hydrophobic, hydrogen bonding -Ni-NTA column (stationary phase) used for today's lan: nitriloacetic acid (NTA) chelates (forms coordinates bonds with Ni2+ ions in the column -Ni2+ binds to His residues which will be the protein of interest (lactate dehydrogenase)
gel filtration chromatography (sep of proteins part 1)
-we will use salt concentration to elute out the molecules instead of changing the pH -the Na+ ion will displace the protein and cause it to elute out of the column
"Parts" Dilution
1:X - 1 part stock: X parts diluent 1:1 = 1 part stock + 1 part diluent 1:9 = 1 part stock + 9 parts diluent 1/X - 1 part stock/X parts total - Similar to Fold dilution 1/2 dilution = 1 part stock + 1 part diluent =1:1 1/10 dilution = 1 part stock + 9 parts diluent = 1:9
How many pKa values would you predict for the amino acid aspartic acid?
3
Which of the following statements below is correct:
40uL of 1N HCl must be added to the pH 10.5 fractions, mix using the vortexer, and then spot fractions on the filter paper strips
HEPES has a pKa of 7.55. At what pH would a HEPES buffer have the maximum buffering capacity?
7.55
mobile phase
=4:1:1 isopropanol: acetic acid: water
beer lambert law
A=ebc -E= extinction coefficient (M^-1cm^-1) -b= length of light path (cm) -c= concentration of a substance (M) -Absorbance (A): -log (T) -Transmittance (T): It/IO
Which of the following statements below is NOT correct?
Affinity chromatography separates proteins based on size
Which statement below is CORRECT with respect to SDS-PAGE?
After adding sample buffer to the protein sample, the proteins will have a net negative charge and migrate towards the positive electrode
In lab this week, you will be given a sample containing a mixture of two proteins: cytochrome c (pI = 9.5) and ovalbumin (pI = 6.0). Cation exchange chromatography will be used to separate cytochrome c and ovalbumin. Which of the following statements below is CORRECT?
At a pH of 6.5, cytochrome c will be positively charged and bind to the negatively charged cation exchange resin
In the alkaline phosphatase assay, you will you be monitoring the formation of product by measuring the absorbance of the solution using a spectrophotometer. Why are you monitoring the formation of product at a pH of 9 and a wavelength of 400nm.
At pH 9, the product, p-nitrophenol, will be ionized, the solution will appear yellow in color, and thus can be monitored at the wavelength of maximum absorption for the phenolate ion which is 400nm.
C1V1=C2V2
C1V1 = C2V2 C1- Initial concentration of stock solution (solution you have) V1- Volume of stock solution to use (what you need to determine) C2- Final concentration wanted V2- Volume of solution you want to make
What type of inhibition is characterized by a change in Km but no change in Vmax?
Competitive
If you would like to determine the concentration of an Unknown solution using a standard curve and the absorbance reading of your Unknown sample falls outside the range of your standard curve (the absorbance reading of your Unknown solution is higher than the most concentrated standard):
Diluted samples of the Unknown solution should be prepared so that the absorbance of the Unknown solution is within the range of the standard curve. The standard curve can then be used to determine the concentration of the diluted Unknown sample. In order to determine the actual concentration of the Unknown solution, you must multiply by the dilution factor that was used to prepare the diluted sample.
precision is determined via standard deviation
E= sum Xi= each score X bar= the mean or average n= the number of values
Which of the statements below is CORRECT?
For the Superoxide Scavenging Assay, you will be provided with 1mL poystyrene cuvettes and for each assay performed, a new cuvette should be used
Given the information below, which of the following statements is CORRECT? Protein Molecular Weight (kDa) pI A. 55 6.0 B 13. 5.9 C. 57 9.6
Given a mixture of Protein B and Protein C, gel filtration chromatography or ion exchange chromatography could be used to separate the two proteins
Bronsted Base
H+ acceptor
ionization of water
H+ does not exist freely in water
Bronsted Acid
H+ donor
Henderson Hasselbalch Equation
HA -> A- + H+ ka= [A][H+]/[HA] pH= -log[H+] pka = - log (ka) pH= pKa + log [A-]/[HA]
You are given a mixture of the following two proteins: Protein 1 (MW 65kDa) and Protein 2 (MW 25kDa). Which of the following statements below is correct?
If the sample is loaded in a well and separated via SDS-PAGE, you would predict Protein 2 to migrate faster
distribution coefficient (Kav)
Kav' = Ve-Vo/Vt-Vo
Components of a spectrophotometer
Light source, monochromators (wavelength selector) sample cell, photodetectors
Which is the weak acid and which is the conjugate base below?
NaH2PO4 (weak acid)+ Na+ -> Na2HPO4 (conjugate base)+ H+
does absorbance have units?
No!
normality
Number of reactive equivalents per liter of a substance
Volume by Volume (% v/v)
Relative to volume of solution, not volume of solvent Eg: 80% v/v CH3OH -> 80 mL CH3OH in 20 mL H2O
Eg: How would you prepare 100mL of 10mM NaCl from a 4M stock?
Step 1: List what you do and do not know. C1 = 4M V1 = Unknown Step 2: Use C1V1 = C2V2 C2 = 10mM V2 = 100mL Note: Units on the left must balance units on the right Add 0.25mL of the stock to the graduated cylinder and add up to 100mL using distilled water
If a beverage enriched with antioxidants is added to the Superoxide Scavenging Assay, which of the following statements below is CORRECT?
The absorbance at 560nm would decrease because the amount of NBT reduced would decrease
accuracy
The degree of closeness of measurements of a quantity to the actual (or true) value.
In the Separation of Proteins Part I lab, absorbance measurements will be used to monitor the elution of dyes and proteins off of the columns. Which statement below is CORRECT?
The elution of cytochrome c will be monitored at a wavelength of 280nm and 400nm, the elution of ovalbumin will be monitored at a wavelength of 280nm, the elution of vitamin B12 will be monitored at a wavelength of 360nm, and the elution of blue dextran will be monitored at a wavelength of 640nm.
In the Separation of Proteins Part III lab, you will be performing the technique of Western Blot. Which statement below is NOT correct?
The same primary antibody will be used for the cytochrome c, ovalbumin, and beta-galactosidase blots
In lab this week, students will be using a spectrophotometer to measure the absorbance of various solutions. Which statement below is correct:
The samples must be placed in the 3mL cuvette from your locker and the cuvette must be placed into the spectrophotometer in a specific orientation to measure the absorbance
In the Buffers and pH lab, you will be using two techniques to identify an unknown amino acid: thin layer chromatography and titration
True
SDS-PAGE separates proteins based on size.
True
Sample buffer must be added to your samples before loading them onto the gel. Sample buffer contains beta-mercaptoethanol which has a strong odor and must be used in the hood.
True
Which of the following statements below is NOT correct regarding thin layer chromatography:
Using pen, write your initials and section # on the plate
In gel filtration chromatography, what dye will be used to indicate the total volume of the column (Vt)?
Vitamin B12 (cyanobalamine)
noncompetitive inhibition (lineweaver burk plot)
Vmax decreases (y-intercept further from 0). Km stays the same (same x-intercept)
competitive inhibition (lineweaver burk plot)
Vmax does not change (same y-intercept). Km increases (x-intercept closer to 0)
From the Lineweaver-Burk plot, which kinetic parameter can be determined from the y-intercept?
Vmaz
Weight by Volume (% w/v)
Weight is measured in grams Volume is measured in milliliters Eg: 5% w/v NaCl -> 5 g NaCl in 100 mL H2O
Which of following statements below is correct?
absorbance values have no units
continuous assay
allows monitoring of product formation as time elapses. Beware! The continuous assay begins when both substrate and enzyme are present together. In our protocol, the moment when you add enzyme to the cuvette is T = 0.
STRONG ACIDS:
dissociate almost entirely in H2O i.e. hydrochloric acid HCl -> H+ + Cl- ka= [H+][Cl-]/[HCl] > 1 -low pkA -weak acid -> conjugate base + H+
Weak acids
don't dissociate as completely in solution i.e. acetic acid -> CH3COOH <-> CH3COO- + H+ ka= [CH3COO-][H+]/ [CH3COOH] < 1 -high pKa
the metric system is ordered by...
factors of 10
How to use a standard curve
graph the known values of concentration correlated with absorbance; use the equation for line of best fit and plug in the absorbance values to get the concentration -cannot extrapolate standard curve (is not always linear) -take unknown 2 and make a 2 or 3 fold dilution -
The pH of a solution with [H+] concentration less than water would be ______________________
greater than 7
A measurement is accurate....
if it measures the true value
The tagged lactate dehydrogenase will be eluted from the column by..................
increasing the concentration of imidazole
vo
initial velocity of the reaction
At a pH of 9.5, would you expect p-nitrophenol to be:
ionized and yellow in color
EDTA
is a chelating agent that sequesters metal ions -deactivates alkaline phosphatase
buffering capacity
is the moles of acid or base required to change the pH by 1
Kcat
kcat: the enzymatic turnover number; the number of substrate molecules an enzyme can convert to product molecules per unit time.
enzyme kinetics plots
lineweaver burk plot vs. michaelis menton
Vmax
maximum velocity
In the affinity chromatography lab which type of interaction will be utilized to purify Lactate Dehydrogenase?
metal ions-histidine containing proteins
Which will travel farther up the TLC plate a nonpolar amino acid or a polar one?
nonpolar
IEC steps
orange part is resin via interaction of amino acid -control via stop cock -tubing will come out of stop cock -first calculate flow rate -lower the meniscus -do not want to dry out column -loading the sample and running it into the column (no disruption) -loading the rest of the buffer -collecting the fractions -labeling the filter paper -spotting the samples on filter paper -special step: pH 10.5 fractions -vortex the fractions -developed filter paper (an hydrin) -keep ALL fractions Until TLC fractions verified -perform TLC on selected fractions
What substrate will be used to assay the activity of alkaline phosphatase?
p-nitrophenyl phosophate
Ion exchange chromatography will be used to separate a mixture of amino acids. The amino acids will be eluted from the column using three buffers: pH 3.5 buffer, pH 6.5 buffer, and pH 10.5 buffer. The buffers must be applied to the column in a specific order. Please select the correct order below:
pH 3.5 buffer first, pH 6.5 buffer second, and pH 10.5 buffer last
protonated
pH < pKa
deprotonated
pH > pKa
amino terminus (N)
pKa= 10
carboxy terminus (c)
pKa=2
stationary phase
polar silica gel
a buffer
resists change in pH
Gel filtration chromatography separates proteins based on____________________.
size
In cation exchange chromatography:
stationary phase is negatively charged and thus will bind positively charged ions
Km
the Michaelis-Menton Constant; an expression of the rate of substrate binding and release AND product release. Not the same as binding affinity. The substrate concentration needed to initiate the reaction.
How does gel electrophoresis work?
the gel is comprised of a gradient of polyacrylamide which helps to separate molecules by their size -larger proteins are trapped by the pores and travel less -smaller proteins fir through the pores and travel further -an electric field is applied to the gel negative at the top and positive at the bottom
In lab you will be using affinity chromatography to purify lactate dehydrogenase from Lactobacillus acidophilus overexpressing lactate dehyrogenase. Which statement below is CORRECT.
the lactate dehydrogenase was tagged with histidine residues to facilitate the interaction with the Ni-NTA column resin
enzyme kinetics
the measure how fast a chemical reaction proceeds under certain conditions (low/high temperature and pH, the presence of activators or inhibitors, etc) -Applications:
The Beer-Lambert Law states that the fraction of light absorbed is proportional to _____________ and _______________.
the path length and the concentration of the solution in which the light passes through
When pH = pKa....
there are equal amounts of weak acid and conjugate base greatest buffer capacity when at the pKa
