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Practical 4 Protein Spectrophotometry

This document provides instructions for a practical experiment on protein spectrophotometry using the Biuret method. It includes a standard BSA concentration series to generate a standard curve, determination of absorbance spectra to identify the optimal wavelength, and questions about the Biuret method. The key steps are to: 1) Generate standard and absorption graphs from the concentration series data. 2) Determine the concentration of unknown BSA solutions using the standard curve. 3) Identify the optimal wavelength for the Biuret method from the absorption spectra.

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Rhulani Makhubela
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67 views5 pages

Practical 4 Protein Spectrophotometry

This document provides instructions for a practical experiment on protein spectrophotometry using the Biuret method. It includes a standard BSA concentration series to generate a standard curve, determination of absorbance spectra to identify the optimal wavelength, and questions about the Biuret method. The key steps are to: 1) Generate standard and absorption graphs from the concentration series data. 2) Determine the concentration of unknown BSA solutions using the standard curve. 3) Identify the optimal wavelength for the Biuret method from the absorption spectra.

Uploaded by

Rhulani Makhubela
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Practical 4: Protein spectrophotometry BCHN 213 2022

[40]

Name and surname:

Student number:

General announcement

Please answer ALL questions and use this template. You will not receive marks for your answers if you do not
use this template. Upload this report to efundi under ‘Assignments’.

No late submissions will be marked.

Use excel to do all the calculations and draw graphs, copy and paste your answers in this

report. Complete table 1. [4]

Table 1: Standard BSA concentration series

Reagent/ Tube number / Buis nommer


Value
Reagens / 1 2 3 4 5 6 7 A B
waarde
0.4 g%
0 0.25 0.5 0.75 1 2 3 0.636 1.344
BSA

0.09 g%
3 2.75 2.5 2.25 2 1 0 2.5 2.5
NaCl
Unknown
/ - - - - - - - 0.5 0.5
Onbekend
A540 nm 0 0.037 0.059 0.088 0.116 0.214 0.261 0.063 0.115
mg BSA 0 1 2 3 4 8 12 0,53 1.12

1
Table 2: Determination of the absorbance spectrum of the reaction mixture for the Biuret method
[3]

Wavelength A Wavelength A Wavelength A

1. Present the results in table 1 and table 2 in graphs


[6]
Note: Draw the graphs on excel. Copy and paste the graphs here.
a.
/ Graph 1: Standard curve for the Biuret method (y-axis: A540 nm; x-axis: [BSA])

2
b.
Graph 2: Absorption spectra of the reaction mixture of the Biuret method (tube 6, table 1).
[4]

3
2. Determine the BSA concentration of the stock solution in the unknown solutions (A and B in table 1).
Remember to add the units. [6]

3. Determine the optimal wavelength (λmax) for the Biuret method from graph 2. [1]
4. If we wanted to measure the BSA absorbance without the Biuret reagent, which wavelength would we
use and why? [2]

5. How will you prepare a 12% BSA solution? Show all your steps.
[2]

6. Name three protein quantification methods learnt during this practical session:
[3]

7. What is indicated by the Biuret test? [1]

4
It is to find the concentrations of proteins and to see if peptide bonds are present.

8. Would the biuret reagent change color if there was only one amino acid in the solution? Motivate
your answer. [2]

9. Explain how the color change occurs with the biuret reagent in the presence of proteins
[2]

10. Match the following items: [4]


1. Reference samples with a A. Wavescan
concentration range
2. Quantity of light of a specific B. Purple
wavelength absorbed by a sample

3. Absorbance values at all C. Absorbance


wavelengths

4. 540 nm D. Standard series

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