(a) 0.25- Turbidity (AU) 0.20- 0.15- 0.10- 0.05- 0.00+ 0 Wt Mutant 1 Mutant 2 Mutant 3 Mutant 4 2 4 8 Incubation time (h) 6 10 Define turbidity. . How is turbidity related to protein aggregation? • Where is protein with no aggregation for any condition? Where is protein with maximal aggregation for any condition? • What area of the plot tells you about the rate of aggregation for any condition? 12 • What area of the plot tells you about total aggregation for any condition? • Slow, medium, and fast rates of aggregation are shown - which samples fall into each category? Two amounts of total aggregation are shown - which

(a) 0.25- Turbidity (AU) 0.20- 0.15- 0.10- 0.05- 0.00+ 0 Wt Mutant 1 Mutant 2 Mutant 3 Mutant 4 2 4 8 Incubation time (h) 6 10 Define turbidity. . How is turbidity related to protein aggregation? • Where is protein with no aggregation for any condition? Where is protein with maximal aggregation for any condition? • What area of the plot tells you about the rate of aggregation for any condition? 12 • What area of the plot tells you about total aggregation for any condition? • Slow, medium, and fast rates of aggregation are shown - which samples fall into each category? Two amounts of total aggregation are shown - which

Human Anatomy & Physiology (11th Edition)

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...

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(a) 1 Normalized fluorescence 0.8 0.6 0.4 0.2 0 50 55 OM 0.100 M 0.200 M 0.300 M 0.500 M 1.00 M 2.00 M 60 113588 65 Temp. (°C) 70 75 80 Where is fully folded protein? • Where is fully unfolded protein? • Where is partially folded protein? • To what does SYPRO orange bind? • Why does fluorescence increase as a function of temperature? ● Define a melting temperature for a protein. • Demonstrate how an estimated melting temperature of the protein in zero molar ligand can be determined. • What is the effect of increasing the molar concentration on melting temperature for this protein? • Why is melting temperature a useful measurement to make for a protein especially if you are interested in protein aggregation?
Protein concentration can readily be determined using the Beer-Lambert law: A = e l c where A = absorbance e = molar absorption coefficient (M-1cm-1) l = light path length (cm) c = concentration (M) If the molar absorption coefficient at 280 nm for yeast ADH is 48860 M-1cm-1 and a 10 mL solution of the protein has an absorbance at 280 nm of 0.4 (as measured by a spectrometer with pathlength 1 cm), then what is the concentration of the protein solution (in μM)? i.e. concentration = ______ μM If the molecular weight of the protein is 36849, what is its concentration in mg/mL? i.e. concentration = _______ mg/mL For each part of the question, show your calculations to arrive at your answers.
(a) 1 Normalized fluorescence 0.8 0.6 0.4 0.2 0 1.7.0- 50 55 OM 0.100 M 0.200 M 0.300 M 0.500 M 1.00 M 2.00 M Where is fully folded protein? • Where is fully unfolded protein? • Where is partially folded protein? 60 A 65 Temp. (°C) 70 75 80 To what does SYPRO orange bind? • Why does fluorescence increase as a function of temperature? • Define a melting temperature for a protein. • Demonstrate how an estimated melting temperature of the protein in zero molar ligand can be determined. • What is the effect of increasing the molar concentration on melting temperature for this protein? Why is melting temperature a useful measurement to make for a protein especially if you are interested in protein aggregation?
The following proteins were separated by SDS-PAGE in the presence of mercaptoethanol. Sketch the relative positions of the various polypeptides on the gel. Label the positive and negative ends of the gel.Protein A: 40 kDa single polypeptideProtein B: 80 kDa protein, made up of two subunits of molecular weight 20 kDa and 60 kDa, held together by noncovalent interactionsProtein C: 200 kDa protein, made up of four identical subunits (50 kDa each) linked together by disulfide bonds
. A protein gives, under conditions of buffer composition, pH, and tem- perature that are close to physiological conditions, a molecular weight by sedimentation equilibrium measurements of 140,000 g/mol. When the same protein is studied by SDS gel electrophoresis in the absence or presence of the reducing agent B-mercaptoethanol (BME), the pat- terns seen, respectively, in lanes A and B are observed. Lane C contains standards of molecular weight indicated. From these data, describe the native protein, in terms of the kinds of subunits present, the stoi- chiometry of subunits, and the kinds of bonding (covalent, noncova- lent) existing between subunits. / A B - BME + BME STD Serum albumin, 67,000 Ovalbumin, 43,000 Carbonic anhydrase, 30,000 Trypsin inhibitor, 20,000 10 2. 4. Co cm migrated
On an SDS-gel, If the distance traveled by the bromophenol blue dye is 7 cm, and the distance traveled by the protein band is 2.8 cm, the mobility of the protein is 40 4 40% 0.4
A sample mixture consists of three proteins with the following properties: Protein A MW (kDa) 200 Amino acid composition 40% nonpolar, 60% polar B 45 20% nonpolar, 80% polar C 98 85% nonpolar, 15% polar IpH 9 3 5 1. If the mixture is subjected to ammonium sulfate precipitation, which protein will precipitate out first? [Select] 2. If the mixture is subjected to isoelectric focusing, which protein will stop moving nearest to the positive electrode? [Select] 3. If the mixture is subjected to cation-exchange chromatography using a buffer at pH 7, which protein will bind to the resin? [Select] 4. If the mixture is subjected to SDS-PAGE, which protein will be at the bottommost portion of the gel? [Select] * Previous Nex
Consider the following protein mixture: Protein A B C D Molecular Weight (kDa) 50 150 200 350 Affinity to Metal ion === Zn²+ === 1. Using hydrophobic interaction chromatography, the protein that will be eluted last is [Select] 2. Using affinity chromatography, the protein that will be eluted last in a Zn²+-containing column is 3. The protein with the fastest migration towards the anode in SDS-PAGE is [Select] IpH value 7 3 9 5 [Select] [Select] 4. Using a buffer solution with a pH of 4, the protein that will bind to an anion exchanger is 5. The protein that will be eluted last in a gel filtration column is [Select] 6. Using isoelectric focusing, the protein that will have a protein band nearest to the cathode (negative electrode) is [Select] % Hydrophobicity 20 45 75 55
The extinction coefficient or absorptivity (ɛ) of protein A at 340 nm is 6440 M-1 cm-1, whereas protein B does not absorb at 340 nm. What absorbance will be observed when light at 340 nm passes through a 5 mm cuvette containing 10 µM of protein A and 10 µM of protein B? Beer-Lambert-law; A = ɛ x C x1; A = absorbance, C= concentration, 1= pathlength).
The A280 of a protein sample loaded onto a gel was determined to be 0.767 (1.00 cm path length, after subtracting the blank). The total volume of this sample was 428 µL. 19.0 µL of this protein sample was mixed with 19.0 µL of 2X laemalli sample buffer and then 12.0 µL of the entire sample was loaded into the gel and electrophoresed. Calculate the amount of protein that was loaded into the gel (in µg).
How many copies of a protein need to be present in a cell in order for it to be visible as a band on an SDS gel? Assume that you can load 100 µg of cell extract onto a gel and that you can detect 10 ng in a single band by sil ver staining the gel. The concentration of protein in cells is about 200 mg/mL, and a typical mammalian cell has a volume of about 1000 μm³ and a typical bacterium a vol ume of about 1 µm³. Given these parameters, calculate the number of copies of a 120-kd protein that would need to be present in a mammalian cell and in a bacterium in order to give a detectable band on a gel. You might try an order-of-magnitude guess before you make the calcula tions.
A series of standard proteins and an unknown enzyme were studied by gel filtration on a Sephadex G200 (the 200 refers to the maximum pore size in kDa) column. The elution volume Vel for each protein is given in Table 1 below. (a) Plot the data in the form of log Mr versus elution volume. From the line of best fit through the points for the standards, determine the Mr of the unknown enzyme. Explain why ferritin and ovomucoid behave anomalously. Table 1 - The Vel versus Mr data Protein Mr Vel (mL) Blue dextran* Lysozyme Chymotrypsin Ovalbumin Serum albumin Aldolase Urease Ferritin# Ovomucoid# Unknown 1,000,000 14,000 25,000 45,000 68,500 150,000 500,000 700,000 28,000 ⎯ 85 200 190 170 150 125 90 92 160 139 *Blue dextran is not a protein but a high-Mr carbohydrate that has a covalently bound blue dye, and it elutes with the void volume of the column. # Do not…