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13 Dec 2019

Isolation and Characterization of the Enzyme Alkaline Phosphatase from Escherichia coli: Protein Quantitation lab

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(make data up ) Using the absorbance data from 280nm and 260nm, calculate the concentration of AP in your fraction. How does this value compare to the value obtained using only the absorbance from 280nm.

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5.3. For another research project, you have labeled a protein with two different dyes made by ATTO-TEC GmbH, a German company that makes some very nice dyes as alternatives to the Alexafluor series that was discussed in class. For this experiment, you have used the ATTO-520 and ATTO-633 dyes.

You have completed the labeling reaction, purified the doubly-labeled protein so that there is not âfreeâ dye in the solution (i.e. all dye is covalently attached to the protein), and obtained the absorbance data below. From these data you can calculate the concentration of each dye in the solution and the concentration of the protein itself (e_280nm = 72,560 M^-1 cm^-1). Using those numbers, you now need to calculate the labeling efficiency from your reaction, i.e. what fraction of the protein molecules have been modified by each of the two dyes.

This is, in essence, a straightforward Beerâs Law absorbance vs concentration question, but there is an added complication that the two dyes have some absorbance at 280 nm in addition to their main absorbance peaks in the visible region of the spectrum. You will therefore need to find both the extinction coefficients of the dyes, which you can use to calculate their concentrations, and the Correction Factor you need to account for how much dye absorbance there is at 280 nm. In effect, the observed absorbance at 280 nm is the sum of the absorbance from the protein and both of the dyes, and you need to find the protein only absorbance before you can calculate the real protein concentration.

To solve this you need to check out the ATTO-Tec web site at www.atto-tec.com where the Support section site has a document called Data-Table that will be extremely useful for answering the question.

Wavelength Absorbance

280 nm 1.31

517 nm 1.32

630 nm 2.21

Can you please help post lab question..

DATA AND CALCULATIONS

Absorbance

Trial 1

0.039

Trial 2

0.045

Trial 3

0.063

Trial 4

0.086

Absorbance of standard (Trial 5)

0.792

Temperature

25Â°C

K_c expression K_c =

[Fe³⁺]i

Trial 1

Trial 2

Trial 3

Trial 4

[SCN^â]_i

[FeSCN²⁺]_eq

[Fe³⁺]_eq

[SCN^â]_eq

K_c value

Average of K_c values

Kc = ________________________ at ___25_____Â°C

beaker	Fe(NO₃)₃ (mL)	KSCN (mL)	H₂O (mL)
1	5	2	93
2	5	3	92
3	5	4	91
4	5	5	90

PROCESSING THE DATA
1. Write the Kc expression for the reaction in the Data and Calculation table.
2. Calculate the initial concentration of Fe3+, based on the dilution that results from addingKSCN solution and water to the original 0.0020 M Fe(NO3)3 solution. See Step 2 of theprocedure for the volume of each substance used in Trials 1-4. Calculate [Fe3+]i using theequation:

[Fe3+]i =

Fe(NO3)3 mLtotal mL X (0.0020 M)

This should be the same for all four test tubes.
3. Calculate the initial concentration of SCNâ, based on its dilution by Fe(NO3)3 and water:

[SCNâ]i =

KSCN mLtotal mL X (0.0020 M)

In beaker 1, [SCNâ]i = (2 mL / 100 mL)(.0020 M) = .000040 M. Calculate this for the otherthree beakers.
4. [FeSCN2+]eq is calculated using the formula:

[FeSCN2+]eq =

AeqAstd

X [FeSCN2+]std

where Aeq and Astd are the absorbance values for the equilibrium and standard solutions,
respectively, and [FeSCN2+]std = (1/100)(0.0020) = 0.000020 M. Calculate [FeSCN2+]eq foreach of the four trials.
5. [Fe3+]eq: Calculate the concentration of Fe3+ at equilibrium for Trials 1-4 using the equation:

[Fe3+]eq = [Fe3+]i â [FeSCN2+]eq

6. [SCNâ]eq: Calculate the concentration of SCN- at equilibrium for Trials 1-4 using theequation:

[SCNâ]eq = [SCNâ]i â [FeSCN2+]eq

7. Calculate Kc for Trials 1-4. Be sure to show the Kc expression and the values substituted infor each of these calculations.
8. Using your four calculated Kc values, determine an average value for Kc.

please do all part :(

1. Be sure to include all the calculations and comparisons requested during the in-lab portion of the experiment.

a. Initial [HOAc]: Show your calculation

b. Calculate KD after simple extraction with diethyl ether, KD = Show your calculation

c. Calculate KD after simple extraction with dicholoromethane, KD = Show your calculation

d. Based on the calculated KD values in parts b and c, explain which solvent was more efficient at extracting HOAc in terms of the intermolecular forces (H-bonding, dipoledipole, Van der Waals, ion paring) of attraction between the solvent and HOAc.