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Laboratory Techniques and Protein Purification, Exams of Biochemistry

A.T. Still University of Health Sciences (ATSU)Biochemistry

A comprehensive guide on various laboratory techniques, including dilutions, calculations, and graph interpretation. It also covers protein purification methods, such as column chromatography and sds-page, and their underlying principles. Information on protein expression, quantification, and purity, as well as antibody nomenclature and western blot development.

Typology: Exams

2023/2024

Available from 05/30/2024

DrShirleyAurora
DrShirleyAurora 🇺🇸

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Biochem Lab Final
1. Be able to use the concept of ratios and proportions to solve a common laboratory calculation. -
Amount: single unit that describes a number, mass/weight, or volume.
Concentration: ratio that describes that describes an amount/total volume.
2. Be able to use the concept of percents to solve a common laboratory calculation. -
% w/v = ? g/100 mL
% v/v = ? mL/100 mL
3. Be able to use the concept of dilutions to solve a common laboratory procedure like single dilutions,
multi-component dilutions, independent dilution series (step dilutions), or serial dilutions. -
Independent dilution series (step dilutions): there are different dilution factors within the
series.
4. Be able to solve common laboratory calculations involving concentrations that are expressed as %
weight per volume, % volume per volume, weight per volume, molarity, and "X". -
1X: working solution
10X: stock solution
5. Be able to accurately make a molar solution from a dry chemical stock. -
M = moles/L = (g/MW)/L = (g/g/mole)/L
6. Be able to accurately make a dilution of a stock solution using either a pipetman or a pipet aid. -
Formula: C1V1 = C2V2v
7. Be able to accurately make a multi-component solution using either a pipetman or a pipet aid -
Several dilutions are combined together to make one mixture from several different stock
solutions.
8. Be able to accurately perform a serial dilution using either a pipetman or a pipet aid. -
All the solutions are diluted by the same factor.
9. Know and apply the common concepts to avoid error while using pipetman and laboratory balances.
-
P20: 2-20 uL
P200: 20-200 uL
P1000: 200-1000 uL
10. Given a variety of common laboratory glassware, utilize the most accurate when preparing
solutions. -
Large volumes: Graduated Cylinder
Small volumes: Pipette
Very small volumes: Micropipette
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Partial preview of the text

Download Laboratory Techniques and Protein Purification and more Exams Biochemistry in PDF only on Docsity!

Biochem Lab Final

  1. Be able to use the concept of ratios and proportions to solve a common laboratory calculation. - Amount: single unit that describes a number, mass/weight, or volume. Concentration: ratio that describes that describes an amount/total volume.
  2. Be able to use the concept of percents to solve a common laboratory calculation. - % w/v =? g/100 mL % v/v =? mL/100 mL
  3. Be able to use the concept of dilutions to solve a common laboratory procedure like single dilutions, multi-component dilutions, independent dilution series (step dilutions), or serial dilutions. - Independent dilution series (step dilutions): there are different dilution factors within the series.
  4. Be able to solve common laboratory calculations involving concentrations that are expressed as % weight per volume, % volume per volume, weight per volume, molarity, and "X". - 1X: working solution 10X: stock solution
  5. Be able to accurately make a molar solution from a dry chemical stock. - M = moles/L = (g/MW)/L = (g/g/mole)/L
  6. Be able to accurately make a dilution of a stock solution using either a pipetman or a pipet aid. - Formula: C1V1 = C2V2v
  7. Be able to accurately make a multi-component solution using either a pipetman or a pipet aid - Several dilutions are combined together to make one mixture from several different stock solutions.
  8. Be able to accurately perform a serial dilution using either a pipetman or a pipet aid. - All the solutions are diluted by the same factor.
  9. Know and apply the common concepts to avoid error while using pipetman and laboratory balances.
  • P20: 2-20 uL P200: 20-200 uL P1000: 200-1000 uL
  1. Given a variety of common laboratory glassware, utilize the most accurate when preparing solutions. - Large volumes: Graduated Cylinder Small volumes: Pipette Very small volumes: Micropipette
  1. Know and be able to apply the differences between the terms: accuracy, precision, standard deviation, and reproducibility. - Accuracy: correctness of the data Precision: variability of the data Reproducibility: variability observed when the entire experiment is performed again. Standard Deviation: quantitative measurement of precision and reproducibility.
  2. Know graphically and statistically what standard deviation means and how it relates to precision and reproducibility. - If the standard deviation is high, then the variability will be high so the precision and reproducibility will not be good because the numbers are off.
  3. Be able to calculate the average and the standard deviation for a set of data. -
  4. What is the physiological role of phosphatases? - Removes phosphate groups Synthesizes ATP and nucleotides Synthesizes phospholipids Regulates metabolic pathways
  5. What are the two main groups of phosphatases and how are they classified? - Phosphatases are classified based upon their pH optima:
  6. Acidic
  7. Alkaline
  8. What is the basis of the reaction to detect the presence of phosphatase activity? - Enzyme activity must be stopped by denaturing the enzyme to alter the structure of phenolphthalein to turn into a pink color. Darker pink = more activity.
  9. What is the purpose of the sodium carbonate? - Stops the reaction by increasing the pH and alters the shape of Phenolathein causing it to turn pink.
  10. Be able to interpret graphs that depict phosphatase activity with respect to changes in pH, temperature, and reaction time. - Changes in pH, temperature, or reaction time can cause changes in protein structure and consequently function.
  11. Why is it important to considering the application of a purified protein prior to actually designing a purification scheme? - Weight, purity, activity, yield, time, and cost must considered before purifying a protein to create the best purification scheme possible.
  12. What are the four intellectual steps involved in optimizing the purification of a protein? -
    1. Development of a suitable assay
  13. Selection of the best source material
  14. Solubilization of the desired protein
  15. Development of a suitable series of fractionation steps
  1. What is the purpose of adding ampicillin and chloramphenicol to the media? - To increase the selection of the pLysS plasmid by killing the sensitive bacteria.
  2. Why do we take culture samples before and after the addition of IPTG? - To indicate how much rGFP was expressed with the effects of IPTG addition.
  3. Who first discovered GFP? - Osamu Shimomura (1962)
  4. Who first cloned GFP? - Douglas Prasher (1992)
  5. What was the first transgenic organism to express GFP? - E. coli (1994)
  6. What was the first transgenic eukaryote to express GFP? - C. elegans (1994)
  7. What is the underlying principle that allows for the purification of rGFP? - Exploiting the physical differences (shape, charge, hydrophobicity, and physical size) between your molecule of interest and all other molecules.
  8. Why does freezing and then thawing the bacterial pellet result in lysis? - The cells slowly freeze so that the cell walls will puncture and quickly thaw to burst open so the chain of lysis will begin.
  9. What is the purpose of the "wash" step when developing the column? - To remove any contaminating proteins that don't bind to the column using the breaking buffer.
  10. How are we monitoring the presence of rGFP? - Qualitative: hand-held UV light Quantitative: fluorescent plate reader readings
  11. If we were purifying a histidine tagged phosphatase, how would we monitor its presence during the purification procedure? - Phenolphthalein can be used to detect the presence of phosphatase in the solution.
  12. What is the underlying principle that allows for the elution of rGFP from the Ni+2-agarose column?
  • Chromatography separates molecules based upon the differential interaction with both a mobile phase and a stationary phase
  1. What are the five major types of column chromatography and the underlying scientific principles that make them useful for separating proteins? -
    1. Gel filtration chromatography (size exclusion): size or molecular weight
  2. Ion-exchange chromatography (anion or cation exchanger): charge-charge interaction
  3. Affinity chromatography: specific molecular binding properties
  1. Hydrophobic Interaction Chromatography (HIC): difference in hydrophobic properties
  2. Partition chromatography: polarity or water solubility
  3. Based upon the design and principle of spectrophotometry, what improper laboratory techniques could result in incorrect absorbance readings? - Scratches on cuvette surface = reflection Air bubbles/bacteria turbidity = scattering Oil fingerprints on cuvette surface = absorption
  4. Given the conditions of an enzyme assay, be able to describe what components of that assay should be placed in the reference cuvette to properly "blank" the machine. - The blank solution would contain everything except what's being measured.
  5. Be able to understand and correctly use the following terminology: wavelength, wavelength scan, maximum wavelength, absorbance, and transmittance. - Wavelength: distance between crests of a wave. Wavelength scan: measures the absorbance of the sample. Maximum wavelength: wavelength of the highest absorbance peak Absorbance: light that stays absorbed in the sample. Transmittance: light that comes out of the sample.
  6. What are the principle differences between a spectrophotometer and a spectrofluorometer? - Spectrophotometer: measures the absorbance of the sample. Spectrofluorometer: measures the fluorescence of the sample.
  7. Given the necessary data, construct and use a standard curve to identify the amount and concentration of an unknown protein sample. - Couldn't upload my own image. Trace from measured to best fit line to concentration
  8. Define specific activity. - The ratio of the enzymatic activity to the total protein amount in a sample.
  9. What are the two common procedures for quantifying protein concentration that we used in this week's laboratory? - Absorbance at 280 and 260 nm Bradford Method
  10. What are the underlying principles of each of those procedures? - Absorbance at 280 and 260 nm: The concentration will be determined using the amino acids Tryptophan (W), Tyrosine (Y), Phenylalanine (F). Bradford Method: after preparing a BSA standard curve, the Bradford dye is added to the samples and absorbance is measured at 595 nm.
  11. What are the advantages and disadvantages of the 280/260 assay and Bradford? - Absorbance at 280 and 260 nm: although this method is rapid and doesn't destroy the sample, it's inaccurate unless the protein is pure. Bradford Method: although this method is sensitive and accurate, the Bradford dye stains the cuvette and is difficult to remove.
  1. What are the three types of membranes discussed in class? Why is one chosen over another? - Nitrocellulose: low tensile strength, low protein binding capacity, and convenient to use. PVDF (PolyVinylidene DiFluoride): high chemical resistance and tensile strength, high protein binding and retentive capacity, and can be "handled" repeatedly. Nylon: Can be "handled" (stripped and reprobed) repeatedly.
  2. What is the purpose of incubating the membrane with BSA, gelatin, or a mixture of dry milk? - To saturate all the sites on the membrane not already bound with the protein.
  3. What is the difference between a primary antibody and a secondary antibody? - Primary antibody links to the protein of interest directly, while secondary antibody binds to the primary antibody (that is linked to the POI) and is conjugated with an enzyme.
  4. What is the purpose of the Tween 20 used in the wash steps? - To disrupt the hydrophobic interactions between the primary antibody and other proteins on the membrane that formed during the incubation.
  5. What is the advantage of using a colorimetric enzyme (like horse radish peroxidase) conjugated to a secondary antibody as opposed to being conjugated to the primary antibody? - Binding will occur and signal detection will be amplified.
  6. What are the advantages and disadvantages of using a chemiluminescent reaction versus a colored precipitate reaction. - Chemiluminescent reaction is expensive yet sensitive while colored precipitate reaction is inexpensive yet less sensitive.
  7. Know and understand how antibodies are "named." - Secondary antibody: Host, antigen, and detection method Primary antibody: Host and the main antigen
  8. Given several antibodies, determine which ones would be used in which order to develop a western blot. - Primary antibody: Mouse anti-Xpress Epitope MAb Secondary antibody: Sheep anti-mouse IgG conjugated horse radish peroxidase polyclonal antiserum solution
  9. What is the difference between monoclonal and polyclonal antibodies? - Monoclonal antibodies are made from identical cells and only recognize 1 epitope while polyclonal antibodies are made from different cells and recognize multiple epitopes. Define Epitope - Epitope: a sequence of 8-10 amino acids physically recognized by an antibody. Define Antigen - Antigen: a foreign particle that elicits an immune response. Define Antibody -

Antibody: a protein generated by an immune system in reaction to a foreign substance. Define Antiserum - Antiserum: antibody of interest is present. Define Serum - Serum: antibody of interest is absent. Define Pre-immune Serum - Pre-immune serum: serum extracted prior to immunization. Define Cross-reactivity - Cross reactivity: when the primary antibody binds to the wrong protein (false positive)

  1. Be able to interpret data given an appropriate figure of coomassie blue stained SDS PAGE gel and a developed western blot. (identify contaminants, degradation products etc.) - The Western blot can identify the C-terminus and contaminants of the protein
  2. Explain why it is important for scientists to study enzyme kinetics. - To determine the efficiency of pharmaceutical inhibitors To see if the two different enzymes utilize similar mechanisms. To determine the order of substrate addition and product release.
  3. What factors can influence enzyme catalyzed reactions? - Enzyme concentration Substrate concentration Temperature pH Ionic strength Cofactor concentration Inhibitor/activator concentrations
  4. What is the difference between a continuous time course assay and a fixed time course assay? - A continuous time course assay measures the rate of reaction in one interval while a fixed time course assay measures the reaction rate of samples at different intervals
  5. Under what conditions would a fixed time course assay be inappropriate? - When the ionic strength of the enzyme is unknown.
  6. What are the assumptions of the Michaelis Menten equation? - [E] remains constant. [ES] formation is rapid and remains constant [S] >>> [E] The reaction proceeds in one direction Define Vmax - Vmax: rate of reaction at full speed

Sandwich ELISA is different from Western blotting, but can be used as a pregnancy test to detect hCG, which functions during the first three trimesters.