Assignment #4 Solution (2)
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Therapeu�cs Engineering CHE 380/580 Assignment #
4
Due 2
/
6
/24, 11 am on BBlearn Name: 1.
You generate a pep�de with the sequence KSLWPWKY. You purify it with HPLC and want to determine how much you made. A�er dissolving it in 1 mL of pure water, you read it on a spectrophotometer in a 0.3 cm diameter cuvete. You get the following reading
(shown as trace A on this graph). a.
What is the pep�de concentra�on?
The ex�nc�on coefficient is 12660 cm
-1 M-
1 (from two tryptophans and one tyrosine). Using beer’s law and the cuvete size of 0.3 cm and es�ma�ng the absorp�on at 280 nm of 1.8, we can calculate 1.8= (12660)*(0.3)* C C= 4.7 x10
-4 M or 0.47 mM. b.
If the ideal yield was 1 mg, what was the efficiency of the reac�on?
Calcula�ng a molecular weight of 1107 g/mol, using 1 mL of water, we calculate we have a total of 0.47 uMols of pep�de, which comes to 0.52 mg, amoun�ng to a 52% yield.
2. From the example ESI
-
MS spectrum, can you
iden�fy at least one
peak tha
t corresponds to an exact mass of:
(Show your reasoning)
a)
1201.77 Da?
401.596 is a triple charged product peak (410.596
-
1)*3= 1201.77
b)
1206.09
Da?
626.0353 is a double charge product with two sodium adducts (626.0353
- 22.99) *2 = 1206.09
3. What is the difference between Disassocia�on constant (Kd) and Effec�ve Dose 50 (EC50)?
Kd describes the concentra�on of the ligand required for binding, while EC50 is the concentra�on required for a physiological effect
4. Use the following affinity plot to answer the following ques�ons:
a)
Name at least two methods that could be used to obtain this plot
See notes: SPR, AUC, etc.
b)
Using this plot, what is the Kd of GST
-
2/3/A binding to Arp 2/3?
Approximately 5 uM
c)
Suppose there is another protein
(Arp 4/5)
that bind
Arp 2/3 with a Kd of 1 uM. Which protein binds �ghter to Arp2/3?
1 uM is a lower Kd and a �ghter binder, meaning Arp 4/5 binds �ghter
5. Describe the steps of whole transcriptome sequencing using standard NGS.
RNA Isola�on, RNA Quality Control, RNA Fragmenta�on, cDNA Synthesis, Second
-
Strand Synthesis, End Repair and A
-
Tailing, Adapter Liga�on, Library Amplifica�on, Library QC, Sequencing and Data analysis
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2. A linear standard curve of KMnO4 is prepared from a set of standard solutions by plotting
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experiment), or Google Sheets/Excel, to plot this standard curve using the data in Table 1. Help with
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Table 1: Data for the standard curve of KMnO4
Absorbance Concentration (M)
0.372
6.00 x 10-4
0.298
0.223
0.149
0.0744
After plotting the standard curve, enter its slope below (not the equation of the line). Report your
answer to 3 significant figures. If your answer ends in one or more zeros, use scientific notation.
Slope =
4.80 x 10-4
3.60 x 10-4
2.40 x 10-4
1.20 x 10-4
M-1
M
3. Suppose that an unknown sample is analyzed spectrophotometrically at the same wavelength that
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50
Percentage
Red No. 40 Soln
100%
80%
60%
40%
Table 4.1: Dilutions of Red No. 40 & Absorbance
Volume of Red No. 40
Soln (mL)
10me
8mL
опи
4mL
Percentage
Red No. 40 Soln
100%
80%
60%
40%
20%
Volume of DI Water
(mL)
OML
2mL
LIML
M
UML
8ML
Table 4.2: Dilutions of Red No. 40 & Absorbance
Concentration Red No. 40
2. Report the wavelength with the maximum absorbance of Red No. 40 dye.
Absorbance
20%
2ML
Part A: Making a Calibration Curve for Beer's Law
1. Calculate the concentrations of each of your diluted Red No. 40 solutions and add these to Table 4.2. Show
at least one sample calculation in the space below.
0.251
0.192
Total Volume
0.181
0·119
0.060
(mL)
10 mL
10 mL
10 mL
10 mL
10 mL
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Please answer. Urgent.
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6.
This is the TLC plate for the solvent: Ethanol 50% / ethyl acetate
2.
50%
V is crystal violet, G is bromocresol green and R is methyl Red. Determine the Rf for each one
(Report to 2 # after the decimal)
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#4
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SPECTROSCOPY
1. If the concentration of solute in a solution is high, the Absorbance will
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2. Was the spectroscopy experiment qualitative or quantitative analysis?
3. If we have 12M HCI and we need 3 L of 1M HCI, how much 12 M
should we use?
4. What does Molarity mean?
5. When must safety goggles be worn?
6. If you make a mistake recording data, what must you do?
spec hw.pptx (2/3)
..%
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2
(1) When performing chemical measurements, replicate experiments are often needed. This is to
mitigate
(Systematic or Random) error.
(2) A CHEM 215 student performed 5 measurements of the wt% of SO4²- of a solid power sample. The
results are 8.75%, 8.85%, 8.89%, 8.71%, 8.64%. The standard deviation is 0.10%. What is the confidence
interval for this student's experiment at 95% confidence level. The t values for 95% confidence for
degree of freedom from 1 to 5 are 12.71, 4.303, 3.182, 2.776, 2.571, respectively. Show your work!
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A stock solution of BSA is available (10 mg/ml) from which the protein standards were prepared. Bradford reagent, 290 ul,
was pipetted into a transparent 96-well microplate. 10 ul of the protein dilution was added followed by mixing in the wells.
After 5 min of incubation at room temperature, the plate was read in spectrum mode with the absorbance spectrometer of
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regression equation that related the protein concentration and absorbance. An unknown BSA sample was prepared the
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0.0625
Protein
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