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Run it! Lab Report
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All biochem lab reports from Spring 2020 semester.
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Principles of Biochemistry
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TEST BANK FOR PRINCIPLES OF BIOCHEMISTRY, 4TH EDITION BY ROBERT A HORTON
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Run It! Questions (must be typed)Question 1: Gel Electrophoresis
Q1a. (3 marks)
Explain the difference between SDS-PAGE and PAGE. In your explanation, be sure to
explain why you need SDS for polyacrylamide gel electrophoresis and describe the
advantages of both techniques.
SDS-PAGE separates proteins based on size only whereas PAGE separates proteins based on
size and charge. SDS is necessary for PAGE because it adds a negative charge on all proteins
and allows the proteins to travel towards the anode. SDS can resolve proteins from 200 kDa
and smaller which accommodates the average protein size of 2 to 130 kDa.
PAGE samples are placed in highly cross-linked gel matrix buffered with mild alkaline solution
so that anionic proteins migrate to the anode and separate along the way based on charge and
molecular weight to allow a specific differentiation of the proteins.
Q1b. (1 mark)
Why do we need a stacking gel in SDS-PAGE?
It has a lower pH and concentration of polyacrylamide to allow faster diffusion of protein
molecules for stacking and eventually lead to the resolving gel.
Q1c. (3 marks)
Describe two techniques used to detect proteins in a gel, other than stain free
technology. What is the advantage of each technique?
Coomassie blue staining:
Soak the gel in dye solution containing methanol, acetic acid, and water and then de-stain the
gel electrophoretically to visualize the bands.
Silver staining:
A metallic silver is deposited on the surface at the band location. Silver ions then interact and
bind to select functional groups to identify the protein.
★ Q1d. This question will be marked. You will need reference(s). (2 marks)
Is it possible to run a protein (size of 300 kDa) and BSA (size of 66 kDa) at the same time
with 7.5% polyacrylamide gel? Explain why or why not.
No – two proteins will not be resolved at the same time due to the differences in their molecular
weight. The resolving ability of the 7.5% acrylamide gel lies between 5 to 250kDa which would
not accommodate for a 300 kDa protein (Bio-Rad). Another reason would concern the fact that
proteins with similar molecular weights would allow efficient separation (Langen, Röder,
Juranville, and Fountoulakis, 1997). The wells in the 7.5% polyacrylamide gel is made
specifically for proteins no larger than 200 kDa for efficient resolution. A 300 kDa protein would
require larger pore in the stacking gel to be able to commence movement through the resolving
gel.
Question 2 – Protein Quantification I
Q2a. (2 marks)
Your supervisor wants you to dilute a 1 mL stock BSA solution (1 mg/mL) into 1:10
dilutions.
● What is the final concentration of diluted BSA solution? Show your work.
● How much volume (mL) of BSA and distilled water would you need to perform the
dilution? Show your work.
Final concentration: 1mg/mL x 1/10 = 0.1 mg/mL BSA
Dilution: 1mL BSA/10 = 0.1 mL BSA; therefore, 1mL – 0.1mL = 0.9 mL Water
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