(a) 1Normalized fluorescence0.80.60.40.201.7.0-5055OM0.100 M0.200 M0.300 M0.500 M1.00 M2.00 MWhere is fully folded protein?• Where is fully unfolded protein?• Where is partially folded protein?60A65Temp. (°C)707580To 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 ligandcan be determined.• What is the effect of increasing the molar concentration on melting temperature for thisprotein?Why is melting temperature a useful measurement to make for a protein especially if youare interested in protein aggregation?

SYPRO Orange is a fluorescent molecular probe that binds to hydrophobic surfaces non-specifically…

Answered: (a) 1 Normalized fluorescence 0.8 0.6…

Biology 2e

2nd Edition

ISBN:9781947172517

Author:Matthew Douglas, Jung Choi, Mary Ann Clark

Publisher:Matthew Douglas, Jung Choi, Mary Ann Clark

Chapter4: Cell Structure

Section: Chapter Questions

Problem 43CTQ: Pathogenic E. coil have recently been shown to degrade tight junction proteins during infection. How...

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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?
How many copies of a protein need to be presentin a cell in order for it to be visible as a band on an SDSgel? Assume that you can load 100 μg of cell extract ontoa gel and that you can detect 10 ng in a single band by sil-ver staining the gel. The concentration of protein in cellsis about 200 mg/mL, and a typical mammalian cell has avolume of about 1000 μm3 and a typical bacterium a vol-ume of about 1 μm3. Given these parameters, calculatethe number of copies of a 120-kd protein that would needto be present in a mammalian cell and in a bacterium inorder to give a detectable band on a gel. You might try anorder-of-magnitude guess before you make the calcula-tions.
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.
7. 1 B e here to search Required Study the two diagrams below. Diagram A 2 1 Diagram B 2 Based on the sequence of steps (1, 2, 3), which of the diagrams shows what would happen to proteins made at the ribosomes and transported to the outside of the cell? Diagram A Diagram B Both Diagram A and Diagram B Neither Diagram A nor Diagram B DELL 40)
You have purified Protein 'X' and you want to know its concentration. As we learned, you can calculate concentrations by simply measuring UV280 absorbance of protein solutions. Using a UV spectrophotometer, you measured an absorbance of 0.6. Given that Protein X has an absorptivity (or extinction coefficient) of 0.2 mL•mg-cm at 280 nm, what is the concentration of purified protein solution (assume the light path is 1 cm)? -1 O A. 3 g/mL B. 3 mg/mL OC.0.2mg/mL OD.0.2 g/mL OE. 0.6 g/mL
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
B W S sult, it makes the protein Why is denaturing the proteins necessary for SDS PAGE to work? Use YOUR OWN X mand 3 E D с $ R F 5 Lê 6 T G MacBook Pro B Y H U N J 8 I M 9 K O I O L 44 P command . : 8 ; x { + C option ? I } 1 M
Given the description of four different proteins above: Which protein will have the highest mobility in an SDS-PAGE gel?
Consider the following properties of the protein components of a sample mixture as provided in the table below. Protein Molecular IpH Percentage of polar amino acid residues Weight (kDa) (%) АСЕ 200 7 20 CLU 25 65 DIA 100 10 40 НЕА 50 80
12. 13. 14. 15. 16. 17. 18. 19. 20. What do SDS and DTT in the sample buffer do? What is the purpose of the bromophenol blue? How does the percentage of acrylamide affect mobility? What is the relationship between molecular mass and mobility in the gel? Estimate the molecular mass of the protein using the standard curve Compare the uninduced and induced cell extracts? Compare the induced cell extract and the purified sample? What is the difference between a denaturing gel (as you have just done) and a non-denaturing gel? Can SDS-PAGE be used to determine the mass of a multimeric protein?
(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 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 samples fall into each category? • Name two samples that have the similar rates of aggregation, but different amounts of total aggregation. • Name two samples that have different rates of aggregation, but similar amount of total aggregation. ● Why is protein turbidity a useful experiment to perform for a protein especially if you are interested in protein aggregation?
What is the function of sodium dodecyl sulfate (SDS) in SDS-PAGE? stabilizes the gel matrix, improving resolution during electrophoresis SDS solubilizes proteins to give them uniformly negative charges, so the separation is based purely on size. SDS raises the pH of the gel, separating multiunit proteins into individual subunits. SDS solubilizes proteins to give them uniformly positive charges, so separation is based purely on pH.

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