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Define recombinant DNA technology
Using molecular biology techniques such as cloning, PCR, restriction enzymes digestion, and gel electrophoresis to take a gene from one source joining it with another piece of DNA, creating a new recombinant DNA molecule that can be inserted into bacteria (or other host) and further studied.
Name three characteristics of DNA
5' -> 3' directionality
Base pairing (AT, GC) via hydrogen bonding
What is the central dogma of molecular biology?
Information is only written in one direction
DNA encodes RNA (through transcription) which encodes protein (through translation)
Describe an amino acid. What is meant when one says proteins are translated "N to C"?
An amino acid consists of an amino-terminus, an a-carbon with an R group (variable, 20 total), and a carboxyl-terminus.
Proteins are synthesized by the ribosome "N to C" which means that the first of amino acid (methionine) will have a free N-terminus, and its carboxyl group will form a peptide bond with the amino groups of the next amino acid. The end of the protein will have a free C-terminus.
Describe the four levels of protein structure
- Primary (amino acid sequence)
- Secondary (a-helices and B-pleated sheets)
- Tertiary (overall 3-dimensional folded structure)
- Quaternary (multiple polypeptide chains interacting)
When viewing a DNA or protein structure, which of the following best represents what it would look like if you could "see it"?
A) Mesh representation
B) Ball-and-stick model
C) Cartoon representations (a-helices as spirals and B-pleated sheets as arrows)
D) Molecular surface (solve-accessible surface)
E) a-carbon trace
D) Molecular surface (solve-accessible surface)
(this multiple choice question has been scrambled)
Which branch of government oversees workplace safety?
Occupational Safety and Health Administration (OSHA)
What is an MSDS and what type of information would it have?
Material Safety Data Sheet - it contains all of the information about a chemical (storage, health warnings, safety measures, PPE< proper disposal, flammability, what to do if contacted or swallowed, etc...)
The QBM and Microbiology labs at UCF are BSL _____.
Rank the following in order of overall fit as well as most to least protection from chemicals and pathogens: plastic gloves, no gloves, nitrile gloves, latex gloves.
Why are nitrile gloves often preferred to latex gloves?
- Latex gloves offer the best fir and protection, followed by nitrile gloves, followed by plastic gloves, and no gloves.
- Nitrile gloves are often preferred because people with latex allergies can use them.
Name three types of hazards you might see in a laboratory
Biological, chemical, mechanical
True or False: Needles should be recapped and placed in the sharps container
False, needles should never be recapped as this can lead to an accidental stick. They should always be discarded directly in a sharps container.
What tool would you use to measure and transfer each of the following volumes?
15 ul: micropipette
15 ml: mechanical pipettor (Easypet)
150 ml: Graduated cylinder
True of False: When dispensing liquid from the micropipette you should push down to the first stop.
False: Pipettes should be dispensed by pushing down to the second stop to fully expel any liquid remaining in the tip.
What is the difference between preparative and analytical centrifugation?
- Preparative centrifugation is for the preparation of large amounts of material (proteins for purification, bacteria for lysing).
- Analytical centrifugation involves smaller sample amounts and allows one to determine sample purity, molecular weight, equilibrium constant, thermodynamics, etc...
For a quick spindown you should use a ______.
Small benchtop centrifuge
Why are centrifuges usually refrigerated?
To prevent protein denaturation that can occur at room temperature
Which type of rotor is best for pelleting a sample?
Fixed angle rotor
Why aren't all centrifugation bottles clear?
Clear bottles are less resistant to chemicals and stress
What is the difference between spectroscopy and spectrometry?
- Spectroscopy is the separation of light into its wavelengths
- Spectrometry is the measurement of light as it interacts with matter such as DNA and proteins
Why do researchers often choose to use colorimetry over spectrometry?
Colorimetry involves converting a sample (usually protein) into a colored compound. This allows the measurement of the sample at other wavelengths (such as 595 or 750) that won't be affected by DNA absorbance if the sample is impure. These wavelengths also do not require special quartz or UV compatible cuvettes.
Which part of the spectrophotometer selects light of a specific wavelength?
How do plate readers differ from spectrophotometers?
Plate readers are essentially spectrophotometers that use 96 well plates to measure multiple samples at the same time.
DNA absorbs at various wavelengths, why is 260nm used for DNA quantification?
DNA has a maximum absorbance peak at 260nm
How does transmittance differ from absorbance?
- Transmittance is the amount of light that passes through a sample
- Absorbance is the amount of light absorbed by a sample
- Transmittance and absorbance are related by Beer's Law
A sample has a transmittance of 70%, what is it's absorbance?
-log(0.7) = 0.155 absorbance
A sample has an absorbance of 0.75, what is it's transmittance?
10-0.75 = 0.177 = 17% transmittance
A sample gives an absorbance of 0.35 and has an extinction coefficient of 40,000. What is the concentration in micromolar?
A = ∈bc
0.35 = (40,000)(1)(c)
c = 8.75 x 10-6 M, or 8.75 uM
A protein has 4 tryptophans (∊ = 5500) and 5 tyrosines (∊ = 1490). What is its extinction coefficient?
∈ = 4(5500) + 5(1490)
∈ = 29,450
You determine that the cuvette you used to test the mixed solution was defective, and was actually 11mm in width. Would the corrected absorbance (if rechecked in a proper cuvette) be greater than or lesser than the above absorbance? Why?
If the cuvette path length was 11mm (or 1.1 cm), it's absorbance would be erroneously high, so if you rechecked the solution in a proper cuvette it would give a lower absorbance.
The defective cuvette would have place more solution in the light path than should have been, which would absorb more light and give a higher reading than expected.
You purify a sample of DNA and pipette 10 microliters of the DNA into 90 microliters of water and measure this sample in a spectrophotometer, which gives an absorbance of 0.25. What is the concentration of DNA in the original sample?
A260 1.0 = 50 ug of DNA
50(0.25) = 12.5 ug/ml
Dilution factor of 10X, thus concentration is equal to 125 ug/ml
A sample of DNA has an A260:A280 ration of 1.0. Is it sufficiently pure?
No, there is protein contamination. The A260:A280 ratio much be >1.5 for a sample to be sufficiently pure.
Proteins absorb at 280nm. Why is this not generally used for protein quantification?
To measure the A280 of protein accurately one much use special quartz or UV compatible cuvettes, the sample must be free of DNA contamination, the extinction coefficient must be known, and other methods (such as Bradford or Lowry) are faster, easier, and can be performed in inexpensive plastic cuvettes.
What advantages does the Bradford Method offer over all other protein quantification methods?
The Bradford Methods is very quick and can be performed in just a few seconds, while other methods take 15 minutes or longer to obtain a protein concentration.
How might the amino acid content (i.e. more or less of certain amino acids) affect the Bradford Assay?
The Bradford Assay relies on interactions with certain amino acids, so proteins with more or fewer of these amino acids, would give different concentrations in the assay. This is one of the reasons for the high variability seen in this assay.
What do the Buiret, Lowry, and BCA Method all have in common?
All involve copper binding to peptide bonds.
Why is the Lowry Method better than the Bradford Method for protein quantification?
The Lowry (and BCA) method have less variations between protein samples and are therefore more accurate than the Bradford Method.
Why must a standard curve be used for protein quantification?
The standard curve helps to account for variations (non-linear response) in absorbance between different concentrations of samples.
How does an enzyme affect the equilibrium of a reaction?
It doesn't. AN enzyme only affects the rate at which equilibrium is reached.
How do enzymes increase the rate of a reaction?
Enzymes stabilize the transitions states of substrates and lower their activation energy.
Some reactions in the body are exothermic. What does this mean?
Exothermic reactions release energy and therefore have a negative ΔG and are favorable/spontaneous
How does an enzyme catalyze a reaction that has a positive ΔG?
By coupling the hydrolysis of ATP to an enzyme, conformational changes take place that assist in stabilizing the transition state; or by activating the substrates with ATP to allow the reaction to occur.
Describe an enzyme active site and how it performs its function.
The active site consists of various positively-charged, negatively0charged, hydrophobic, aromatic, large, and small amino acids oriented in a 3-dimensional space. Together these amino acids interact with substrates and promote stabilization of the transition state.
How could you identify potential active site amino acids across various species if you only knew the primary sequence?
Perform a protein sequence alignment and look for homology.
How do the two models of enzyme-substrate interactions differ? Which is more accurate?
The Lock and Key Model states that enzymes are static
The Induced Fit Model states that enzymes undergo conformational changes upon substrate binding. The Induced Fit Model is more likely as it explains the transition state stabilization.
Who do enzymes have a Vmax?
Enzymes are limited by either diffusion of the substrates and products of by the time it takes for them to reset before the next reaction can be performed.
What does Km mean, and how does it relate to Vmax?
Km is a measure of the stability of the enzyme-substrate complex, and is the substrate concentration and 1/2 Vmax
Which type of inhibitor makes the best drug, and why?
Noncompetitive inhibitors do not compete for the enzymes active site, but rather shut the enzyme down at another location. Therefore they cannot be overwhelmed as substrate concentrations build up in the cell.
Why would one opt for the Pyruvate Kinase-Lactate Dehydrogenase Assay over a Kinase Assay to measure ATP directly?
- The Pyruvate Kinase-Lactate Dehydrogenase Assay will couple ATP hydrolysis to NADH oxidation, which can be measured in a spectrophotometer.
- A Kinase Assay often requires the use of radioactive ATP, which requires special facilities and can be harmful.
Does the reaction studied in the CDNB Assay have a negative or positive free energy? Why?
The reaction studied in the CDNB Assay (GSH + CDNB) can proceed on its own in the absence of GST. Therefore the reaction is favorable and has a negative ΔG.
Explain the Kinase Assay in your own words from the Kinase Assay figure.
Hot ATP is mixed with a substrate. 32P is transferred to the substrate in the presence of a potential kinase. After the unused how ATP is washed away the phosphorylation can be detected via autoradiography or a scintillation counter.
Convert between kilodaltons and number of amino acids:
500 amino acids = ______ kDa
75 kDa = _______ amino acids
500 amino acids = ~56 kDa
75 kDa = 675 amino acids
Using dimensional analysis, how many kilometer are in 3.11 miles?
Use 1 inch = 2.54cm
3.11 miles = ~5km
How many microliters are in 1.25ml?
How many milligrams are in 20,450 ng?
How many moles/liter does 75 uM equal?
75 uM = 75 micromoles/liter
Why is it so important that a stock solution be properly mixed and homogenous?
Stock solutions must be homogenous because dilutions made form them, and any fraction of the stock solution that is removed, must have a known concentration.
What is the molarity of 5 grams NaCL in 50ml water?
(5g NaCl)(1mol/58.44g)(1/0.05L) = 1.71 M NaCl
How do you prepare 150ml of 0.5 M Tris-HCl pH 8, 25mM NaCl?
(0.5 M Tris)(0.150 L)(121.14g/mol) = 9.09 g Tris
(0.025M NaCl)(0.150 L)(58.44 g/mol) = 0.22 g NaCl
Add 9.09g Tris and 0.22g NaCl to 120ml dH2O in a beaker, pH to 8, then bring up to 150ml in a graduated cylinder
Hydrochloric acid (Mw = 36.4611) comes in a concentrated form at 38% w/w. It has a density of 1.189 g/ml, so in effect it is at 45.182 g / 100ml. What is the molarity of HCl?
(45.182g/100ml)(1mol/36.4611g)(1000ml/1L) = 12.39 M HCl
How do you prepare 500 ml of 30% v/v ethanol?
500 x 0.3 = 150 ml of ethanol, fill to 500ml with dH2O
How do you prepare 225 ml of 25% w/v NaCl?
0.25 x 225 = 56.25 g NaCl, bring up to 225 ml with dH2O
You have 20ml of a 10% w/v solution of ammonium sulfate. How many grams of the ammonium sulfate are in this solution?
20 x 0.1 = 2 grams ammonium sulfate
You want to run a cesium chloride gradient to purify a sample of DNA in an ultracentrifuge. You wish to fill a 15 ml test tube with five equal layers of 5%, 10%, 15%, 20%, and 25% cesium chloride. How much total cesium chloride will you need?
Multiply their percentages by the desired amount (15 ml) and add them all together
(0.05 x 15) + (0.10 x 15) + (0.15 x 15) + (0.20 x 15) + (0.25 x 15) = 2.25 grams CsCl
The concentration of the hemoglobin monomer (17 kDa) is approximately 15 grams per deciliter of blood. The average human blood volume is 5 L. What is the concentration of the hemoglobin monomer in your body in millimolar?
(15g/1 dL)(10 dL/1L)(1 mol/17000g)(1000 mmol/1mol) = 8.82 mM hemoglobin in the blood
You load 10 ug of 60 kDa protein on a gel. How many copies of this protein are there in the single band you see on this gel?
(10ug)(1mg/1000ug)(1g/1000mg)(1mol/60000g)(6.023x1023/1mol) = 1x1014
How many pounds of the hemoglobin monomer does the average person carry? 1 pound = 454 grams
(15g/dL)(10dL/1L)(1lb/454g)(5L) = 1.65 pounds of hemoglobin per person
A recent study states the optimal concentration of vitamin D (Mw = 384) in the blood is 60nM for those who were in the sun for ~12 hours a weekend. If the average blood volume is 5 L, how many micrograms of vitamin D should optimally be found in the body?
(60nmol/1L)(5L)(384ngrams/1nmol)(1ug/1000ng) = 115 ug Vitamin D in the body
The recommended daily allowance of vitamin D if 600 IU's, which in the case of vitamin D, 1 IU = 0.025 micrograms. How many IU's are there in the answer to the above question?
(115ug)(1 IU/0.025ug) = 4600 IUs
You have a 4 M stock solution of phosphate budder. How do you prepare 75 ml of 0.8 M phosphate buffer from this stock?
4 M x V1 = 0.8 M x 75ml
V1 = 15 ml
Add 15 ml of the 4 M stock solution to a graduated cylinder, bring up to 75 ml with dH2O
You have a 5X stock of sample loading buffer. You plan on mixing this stock with some DNA samples and loading them on a gel. Each well of the gel holds 40 ul. How much sample loading buffer and DNA should you mix to fill up the well?
5X x V1 = 1X x 40 ul
Solve for V1 = 8 ul stock, 32 ul DNA
You have a 25 ml sample of protein at 10 mg/ml. If you concentrate this solution to 5 ml, what is the new concentration?
25ml x 10ml/ml = 5ml x C2
Solve for C2 = 50mg/ml protein
You will be performing a CNDB Assay in lab. In this assay you place 10 ul each of 100 mM CDNB (Mw = 202.55) and 100 mM GSH (Mw = 307.32) into a cuvette, bring the volume to 1 ml and measure their reaction. The product is a dinitrophenyl thioether (Mw = 473.42, ∊ = 9600 M-1 cm-1).
a) What are the final concentrations of both substrates?
b) How many milligrams of each is present?
c) When the spectrophotometer reads at 1.0, what is the concentration of the product? How many milligrams is this?
d) What percent (by total body weight) of reactants have converted to product?
a) 10ul x 100mM = 1000ul x C2, C2 = 1mM
b) (1mmol)(10-3 L)(202.55mg/1mmol) = 0.20255mg CDNB, and (1mmol)(10-3 L)(307.32mg/1mmol) = 0.30732mg GSH
c) A = ebc
1.0 = (9600)(1)(c), c = 0.104mM
(0.104mmol/1L)(10-3 L)(473.42mg/mmol) = 0.04924mg
d) 49.24/(202.55+307.32) = 9.66%
Why should DNA and proteins be kept in buffers and not dH2O?
Placing DNA and proteins directly in water will greatly alter the pH, which can affect DNA and protein structure and function. Buffers resist changes in pH and keep DNA and proteins in stale environments.
Why does Tris not buffer well at pH 5.0?
Buffers work best and resist changes in H near their pKa, and since Tris' pKa=8.06, it buffers best between pH 7.5 to 9.0. At pH 5.9 Tris would be exhausted and unable to take up any more hydrogens.
You are pHing a solution of Tris to pH 8.0 with acetic acid, but accidentaly go to a pH of 7.8. What should you do?
In QBM, throw the sample away and start over.
Why should laboratory notebooks be signed by the researcher and PI frequently?
Signing the book ensures that the experiments were completed when they said they were, and were approved by the PI. It is very important when considering credit for a discovery or filing of a patent.
What are some reasons a research might omit steps from a published protocol?
- The step in the protocol ay be common knowledge (such as the Bradford Assay) and not need an in-depth description
- The journal may limit the size of the methods section so the researcher must trim it down
- The researcher may intend to publish the protocol in a methods journal at a later time
- Researcher may wish to protect their protocols and data to prevent competing laboratories from copying their work (which is an ethics violation)
What format do lab reports follow?
The format of a scientific publication in a research journal
How do journal clubs differ from an invited seminar club?
- Journal clubs or departmental seminars are usually more casual and the topic of the discussion is often a recently published high impact paper.
- An invited seminar series involves an internal or external researcher presenting their research in a large forum, and often the entire institution is invited to attend.
Why do graduate students and posdocs present posters at conferences?
To share their research, get ideas, and make connections in the field
Who funds the majority of biomedical research in the US? What is their source of money?
The National Institute of Health (NIH)
Who determines which scientists receive grant money?
A panel of scientists that read grants and rank them according to impact. Only the top fraction of grants are funded.
Why are more researchers choosing to publish in online journals that have impact factors 10X lower than the top tier science journals?
Online journals are open access which means they may be seen by more people, have faster publication times, less peer review, and cost less to publish in. Also, many researchers believer their government-funded research should be free and accessible to all.
Why are the first and last authorship positions so important?
- The first author is seen as the one who designed the project, performed the experiments, wrote the paper, and is the one referenced in citations (i.e. Smith et. al.). The last author is generally the Principal Investigator who secured the funds for the project and helped develop the goals and design the experiments.
Why are citations important?
Citations validate your statements and tell the reader where they can go to read the peer-reviewed background information on a statement you made.
Is it possible to plagiarize yourself?
You test the effect of UV light on protein structure. You increase the UV does across samples and measure structural changes. What is the dependent variable?
The protein structural changes
You read a headline that says "sitting on the couch longer causes one to eat more cookies". Do you question the wording of this headline in any way?
The headline specifically says sitting on the couch causes one to eat more cookies. You should immediately question whether it's the sitting on the couch that causes eating more cookies or some other factor as there are likely other explanations.
Why might research data be biased?
Researchers are generally looking for positive results, and therefore experiments may be chosen that don't explore all potential explanations or designed to prove the hypothesis. Some researchers may succumb to the pressures of the field and intentionally falsify data.
In a double blind experiment, the doctor does not know which pill is the experimental drug and which is the placebo. How could the experiment still be biased?
The researcher could subconsciously convey which pill is the drug, and doctor could convey that message to the patients taking them. Also, the researcher could bias the data when analyzing it.
Who is credited with developing the experimental method, acid synthesis, distillation, and crystallization?
Who showed DNA was the basis for genes?
Who coined the term "Molecular Biology"?
What representation represents what a protein "actually" looks like in the real world?
Molecular Surface ( or Solvent-Accessible Surface) representations
_____ is a tumor supressing cell
p53 is a tumor supressing cell
Who sets forth the basic rules of research laboratories?
What does an MSDS tell you?
States everything important about a particular substance/chemical that may be used in a lab
What organization regulates research labs that work with possible disease causing microorganisms?
What determines the BSL of a research laboratory?
BSL-1 labs work with minimal potential hazards
BSL-2 work with organisms of moderate potential hazard
BSL-3 work with pathogenic microorganisms
BSL-4 work with pathogenic microorganisms that do not have any treatment options
Who regulates internal oversight of a specific institution?
The Laboratory Safety Officer
Who provides comprehensive health and safety programs for institutions?
The Department of Environmental Health and Safety
For volumes 2L - 25ml you would use a _____, for 25ml - 1ml you would use a ______, for 1ml - 1ul you would use a ______.
For volumes 2L - 25ml you would use a graduated cylinder, for 25ml - 1ml you would use a glass/plastic pipette, for 1ml - 1ul you would use a micropipette.
What are the most common types of glass/plastic pipettes? How are they different?
Mohr and Serological
Mohr has a thinner tip for finer measurements (drops will be smaller)
What type of centrifuge needs to have it's samples measured to be ~0.1 grams within eachother? Which one requires ~0.25 grams?
Ultracentrifuges must be balanced within ~0.1 grams
Large centrifuges must be balanced within ~0.25 grams
Which type of rotor is best for pelleting? Which is best for samples analysis?
Fixed-angle rotors are best for pelleting
Swing-bucket rotors are best for sample analysis
Which type of rotor was used in the Meselson-Stahl Experiement?
Describe the five basic components that are of a spectrophotometer.
1. Light source - will be either tungeston or deuterium
2. Monochromater - selects which wavelength will be measured
3. Prism/Grating - disperses light which will exit a narrow slit (which selects only a small range of dispersed light)
4. Sample Compartment - holds sample
5. Detector - light passes through this and uses phototubes/photomultipliers to covert photons to electrical energy
_______ and ______ are signatures of molecules, and ______ can be used to identify and distinguish particular compounds.
Spectral shapes and absorption intensities are signatures of molecules, and spectral properties can be used to identify and distinguish particular compounds.
Quantization is caused by light interacting with electronic and vibrational modes of molecules
What do the following wavelengths correspond to...
260 - ?
- 260 - DNA absorption/concentration (special cuvettes required)
- 280 - Protein absorption/concentration
- 340 - CDNB Assay, NADH in Pyruvate Kinase/ Lactate Dehydrogenase Assay
- 560 - BCA Assay (protein concentration)
- 595 - Bradford Assay (protein concentration)
- 600 - Bacterial growth in LB Media
- 750 - Lowry and DC Protein Assay (protein concentration
Proteins always start with why amino acid?
What absorbs light at 465 and 665 nm, and has one of the highest molar absorptivities?
For homogenous samples, each successive layer will absorb _____ of the incident of light as the previous layer.
For homogenous samples, each successive layer will absorb the same fraction of the incident of light as the previous layer.
For ______ samples, each successive layer will absorb the same fraction of the incident of light as the previous layer.
For homogenous samples, each successive layer will absorb the same fraction of the incident of light as the previous layer.
What is the "molar extinction coefficient"?
It's units are (L mol-1 cm-1), is also known as the molar absorptivity coefficient.
It is the absorbance of a 1M solution when the path is 1cm.
For DNA and proteins, it is dependent on the # of aromatic or ring structures
What are the molar extinction coefficients for the following:
- Tryptophan - 5500
- Tyrosine - 1490
- Cysteine - 125
What device can measure microliter quantities of a DNA sample?
Transmittance decreases _______ with respect to the concentration of the colored compound and to the light path length
Transmittance decreases exponentially with respect to the concentration of the colored compound and to the light path length
Light absorption is proportional to the number of _______.
Light absorption is proportional to the number of molecules in the path.
What are the advantages and disadvantages of Protein Quantification?
Advantages – reproducible, simple to perform, fast, inexpensive, very useful
Disadvantages – only an estimate of protein concentration, interference from suphydryl derivatives, detergents, carbohydrates, amines, nucleic acids, lipids, salts
Which are the lab methods used for Protein Quantification?
- Bradford Method
- Biuret Method
- Lowry Method (DC Protein Assay)
- BCA Method
The most accurate technique for determining protein concentration is _______
The most accurate technique for determining protein concentration is Amino Acid Sequence
What is the fastest/simplest method for Protein Quantification?
Describe the Bradford Assay
Used the dye Coomassie Brilliant Blue (changes color from red(465) to blue (595) as dye binds to protein)
Dye forms strong noncovalent (van der Waals) complexes with proteins based on the relative number of positive charges on the proteins
Does not detect free amino acids or DNA, or anything less than 3000 Da
Optimal dilution range of 0 - 1.5 mg/ml
What are the advantages and disadvantages of the Bradford Assay?
Advantages - Sensitive, accurate, very fast; Compatible with most Common buffers; Chaotropic reagents (6M guanidine-HCl, 8 M urea, sodium azide); During protein purification, can be used as a quick “flow through” check
Disadvantages - Color response is nonlinear over a wide range of protein concentration, therefore a standard curve should be run with each assay; 2X the variability as copper methods; Some proteins that are insoluble in the acidic dye cannot be assayed; Incompatible with surfactants, detergents; Reagent stains cuvettes
The CB-X Assay is a variation of ______.
The CB-X Assay is a variation of The Bradford Assay.
A centrifuges rate of sedimentation is dependent upon the _________.
A centrifuges rate of sedimentation is dependent upon the applied centrifugal field (g) being directed radially outwards.
A centrifuges Centrifugal Field (g) is determined by what?
The Square of the angular velocity of the rotor, and the radial distance of the particle from the axis of rotation (r, in centimeters)
Preparative centrifuges include _____, while analytical centrifuges include ______.
Preparative: Small bench centrifuges, Large capacity refrigerated centrifuges, High speed refrigerated centrifuges, and Ultracentrifuges
The simplest copper-based technique for Protein Quantification is the _______.
The simplest copper-based technique for Protein Quantification is the Biuret Method.
Briefly describe the Biuret Method
The Biuret Method is a copper-based technique for protein quantification (and is the simplest of them)
- In alkaline conditions, copper(II) binds to the peptide nitrogen of proteins Cupric complex absorbs light at 550 nm
- Since the copper reacts with the peptide bond, there is little interference by free amino acids, and the amino acid composition of the proteins is not very important
- Not very sensitive
- Buffer interference from tris and ammonia
The ______ method is an improvement upon the Biuret Method.
The Lowry Method is an improvement upon the Biuret Method.
Briefly describe the Lowry Method
The Lowry Method is a copper-based technique for protein quantification, and is an improvement upon the Biuret Method
- It is a commonly used method (inexpensive, easy, reproducible, 10-20X more sensitive than A280, 100X more sensitive than Biuret Method)
- Copper binds peptide bonds under basic conditions, Cu2+ -> Cu+Cu+ reacts with the Folin reagent (phosphomolybdic-phosphotungstic reagent) which becomes reduced to blue (750 nm)
- Standard curves are linear only at low protein concentration, therefore a standard curve is run with each assay
- Timing/mixing of reagents with the samples must be precise, reagents unstable
- Sensitive to contaminants (Detergents (Lowry, not DC Protein Assay performed in lab), lipids, sugars, pH)
- Linear range – 0.2 to 1.5 mg/ml depending on kit
Briefly describe the BCA Method
- The BCA Method is a copper-based technique for protein quantification that is similar to Lowry reaction, using the BCA (bicinchoninic acid) reagent instead of Folin
- Is faster and easier, and has more stable reagents
- Cu2+ is reduced to Cu+
- Two molecules of BCA chelate to a copper ion
- Sensitive to contaminants (Carbohydrates, catecholamines, tryptophan, lipids, phenol red, cysteine and tyrosine, impure sucrose glycerol, H2O2, uric acid, iron)
- Low protein to protein variation (15%)
What are the six major classes of enzymes?
How would you calculate the equilibrium constant (Km)?
The equilibrium constant can be calculated by dividing the forward reaction (Ka) by the reverse reaction (Kb).
Free Energy (ΔG) is a measure of ______.
the difference in energy between the substrates and products
Who proposed the Lock and Key Model?
Explain what a Vmax is.
At some concentration of substrates, an enzyme will become saturated and will be catalyzing the reaction at it's maximum possible rate, which is the Vmax
An enzymes rate is its ______, and depends on ______.
An enzymes rate is its inherent velocity, and depends on the concentration of the substrates, temperature, pH, and presence of cofactors.
What is the "Michaelis Constant (Km)"?
- The Km is a value that is equal to the sum of the rates of breakdown of the enzyme-substrate complex over its rate of formation.
- It is a measure of the stability of the ES complex (or the affinity of an enzyme for it's substrate).
- Km is also related to an enzymes initial rate (v)
A high Michaelis Constant (Km) indicates ______, while a low Km indicates ______.
A high Michaelis Constant (Km) indicates weak substrate binding, while a low Km indicates strong substrate binding.
How are Michaelis Constant (Km) and Vmax related?
The Km is the substrate concentration which gives a rate equal to 1/2 of the Vmax
Describe the Hydrolysis of ATP
Hydrolysis of ATP is an energetically favorable reaction written as (ATP + H2O -> ADP + Pi) which is used by enzymes to couple to less energetically favorable reactions (ΔG = -7.3 kcal/mol) and drive them forward
Who proposed the Induced Fit Model of enzymes?
Daniel E. Koshland
Define "Saturation" (as related to enzymes)
Saturation: The rate asymptotically approaches a limiting value, called Vmax, becoming zero-order with respect to substrate concentration (not varying with concentration)
What are the two enzyme assays? Which one is a coupled continuous assay?
The CDNB Assay, and the Pyruvate Kinase-Lactate Dehydrogenase Assay (which is a coupled assay)
What is a coupled assay?
A certain reaction may not use products that can be tracked in a spectrophotometer, so often a coupled assay is performed where the reaction of interest is linked to one that is measurable
The Pyruvate Kinase-Lactate Dehydrogenase Assay is an example of a coupled assay
Describe the "Kinase Assay"
- Radiolabeled ATP (“hot” ATP, with 32P) is used is mixed with a protein kinase (or a lysate) and a substrate that can be phosphorylated (like histone)
- Run sample on gel, develop, a band indicates radioactivity and therefore phosphorylation
- Analyze sample (western, sequencing, scintillator)
- Can be used to study signaling pathways or test inhibitors
What are the seven basic SI units?
What is the common unit of measurement for protein mass?
True or False: The "Kilodalton (kDa)" is an SI unit?
False: The kilodalton is a non-SI unit of measurement
Describe the "Kilodalton"
The Kilodalton is the molecular weight of a protein, and can be calculated by adding up all of the individual molecular weights of a protein's amino acids.
One dalton = one atomic mass unit, or 1/12 the mass of C12
Finish the following conversion factors:
1 dalton = ?
1 kilodalton = ? (daltons)
1 amino acid = ?
1 kilodalton = ? (amino acids)
1 ppm = ?
- 1 dalton = 1 gram/mole
- 1 kilodalton = 1000 daltons
- 1 amino acid = ~112 daltons
- 1 kilodalton = ~9 amino acids
- 1 ppm = 1 mg/L
How would one prepare solutions of known molarity?
Amount needed (grams) = Mw
x desired concentration (M) x volume needed (L)
- Ex: To make 50ml of 0.1 M KBr (Mw = 119 g/mol), do the following...
- 119 x 0.1M x 0.050 L = 0.595 g in 50ml
What is the Mw of Tris and NaCl?
Tris = 121.15 grams/mole
NaCl = 58.44 grams/mole
Describe the following Percent Concentrations:
, both the solute
and the solvent
are expressed in the same physical mass units
(grams, kilograms, etc...)
- Ex: 5g NaCl dissolved in 20ml H2O would be 20% w/w (5/25)
- 5 g NaCl / 20 g H2O = 5/(20+5) = 20% w/w
Describe the following Percent Concentrations:
, both the
solute and the solvent are expressed in the same volume units (liters, milliliters, etc...)
- Ex: How do you make 250ml of 25% v/v glycerol?
- 250ml x 0.64 = 160 ml glycerol
Describe the following Percent Concentrations:
, the amount of solute
is expressed in physical mass units (grams), and the amount of solution
is expressed in volume units (milliliters).
- Ex: How do you make 0.675 L of 25% w/v NaCl?
- 675ml x 0.25 g/ml = 168.75 g NaCl
Many buffers consist of ____.
A weak acid or a weak base and its salt which resist changes in pH
The amount of acid and base needed to produce a buffer with the desired pH can be calculated by using the ______.
pH = pKa + log([base]/[acid])
Buffer's have a range in which their resistance to changes in pH are at a maximum, which is based on the buffer's _____.
Acid dissociation constant (pKa), and is the pH at which the acid in solution equals the base
Describe what a Journal Club/Seminar encompasses
- Present another’s work (i.e. current papers from Nature, Science, Cell, etc.)
- Gives graduate students practice analyzing data and presenting it, and the audience practice at discussing data scientifically
- Brings everyone up to date on the current state of the field
- Brings departments together to discuss the paper and new ideas
Describe what an Invited Seminar Series encompasses
- Internal, external researchers
- Present data to a group of colleagues
- Purpose is mainly for teaching, networking, feedback, promoting an area of research
- Occasionally may host Continuing Education credit
Describe what a Conference/Symposium encompasses
- Gathering of experts in a field, or consortiums (for example, a structural biology conference, a tuberculosis, HIV, or cancer conference)
- Features poster sessions and invited lecturers
Describe what a Poster Session encompasses
- Present data on-campus or a scientific conferenceNetworking, feedback
- Often presented by graduate students or post-docs
Research must apply from grants from either ______ or _____
the National Institute of Health (NIH) or obtain private donations
What does the NIH state about how it funds grants?
The NIH..."funds grants, cooperative agreements, and contracts that support the advancement of fundamental knowledge about the nature and behavior of living systems to meet the NIH mission of extending healthy life and reducing the burdens of illness and disability.”
If a research is using human subjects, animals, or certain harmful samples or pathogens, they may require approval from the ______ before a grant may be approved.
Institutional Review Board (IRB)
What type of grant is the most sought after?
- An R01 (a Research Project Grant), which can provide hundreds of thousands or millions of dollars to a laboratory and is funded for 3-5 years.
- New investigators (scientists early in their careers) are given special consideration
Describe positive and negative controls
Positive Controls are used as the "normal" test and should produce the expected, measurable result(s).
Negative Controls should not be observed, and if they are seen it indicates that the sample or reagents are contaiminated
How might a researcher prevent bias?
Use good controls, statistically analyze data, report all data (both good and bad) and any manipulations thereof, undergo the peer review process and experiment duplication, use double-blind experiments, and hold to scientific rigor.