Biochemistry Lab Techniques Quiz

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Questions and Answers

What is the purpose of boiling the protein samples after adding Laemmli Sample Buffer?

To increase the pH of the sample

To enhance the visibility of the proteins

To eliminate all protease contamination

To denature the proteins effectively (correct)

Which component of the Laemmli Sample Buffer is known to be toxic?

SDS

Coomassie Blue

Destain solution

2-mercaptoethanol (correct)

Why is it important to heat protein samples immediately after adding Laemmli buffer?

To ensure a longer shelf life of the samples

To improve protein binding to the gel

To prevent degradation from proteases that resist SDS denaturation (correct)

To increase the temperature for better protein solubility

What should you do before removing gels from the electrophoretic apparatus?

Power off and remove cables Signup and view all the answers

At which voltage should the gels typically be run during the electrophoresis process?

180 volts Signup and view all the answers

How should the Coomassie Blue staining solution be applied to the gel?

In a thick layer to cover the entire surface Signup and view all the answers

What step must be performed after staining the gel with Coomassie Blue?

Pour off the stain and rinse with deionized water Signup and view all the answers

What is the composition ratio of Laemmli Sample Buffer to the sample volume in the preparation process?

1:4 Signup and view all the answers

What is the primary purpose of SDS-PAGE?

To separate proteins based on their molecular weight Signup and view all the answers

In SDS-PAGE, which component is responsible for denaturing proteins?

Sodium dodecyl sulfate (SDS) Signup and view all the answers

Which direction do proteins migrate during SDS-PAGE?

From cathode to anode Signup and view all the answers

Which of the following is NOT a component of Laemmli Sample Buffer?

Aluminum hydroxide Signup and view all the answers

What role do reducing agents like β-mercaptoethanol play in SDS-PAGE?

Disrupt the structure of proteins Signup and view all the answers

How does SDS ensure consistent migration of proteins during electrophoresis?

By binding to proteins and making them negatively charged Signup and view all the answers

What happens to high molecular weight proteins during SDS-PAGE?

They remain at the back end of the gel Signup and view all the answers

Which type of gel is used as a support medium in SDS-PAGE?

Polyacrylamide gel Signup and view all the answers

Study Notes

MD100 Medical Biochemistry I - Lab Exercise 3: Introduction to SDS PAGE

Course: Medical Biochemistry I
Lab Exercise: Introduction to SDS PAGE - Protein identification and characterization
Semester: Fall 2024
Institution: European University Cyprus, School of Medicine

Objectives

SDS-PAGE laboratory technique: Introduction to the technique
Theoretical background: Principle of SDS-PAGE explained
Part A: Sample preparation – dilutions
Part B: Sample loading – Running the gel
Part C: Gel staining and destaining
Part D: Protein gel analysis – protein identification

Introduction to SDS-PAGE

Technique: Used to separate proteins based on their molecular weight.
Electrophoresis: The separation of macromolecules in an electric field.
SDS (Sodium dodecyl sulfate): Denatures proteins, making them uniformly negatively charged. This allows separation by size.
Principle: Charged molecules migrate to the electrode with the opposite charge when placed in an electric field. SDS binds to proteins, giving them a uniform negative charge. Proteins migrate through the gel toward the positive electrode.
Reducing agents: (e.g., β-mercaptoethanol) cleave disulfide bonds in proteins, disrupting their structure.

The principle of SDS-PAGE

Protein Structure: Proteins initially have a folded structure with positive and negative charges.
Reduction: Reducing agents (β-mercaptoethanol) break the disulfide bonds, unfolding the proteins.
SDS Binding: SDS binds to the unfolded proteins creating a uniform negative charge, proportional to the polypeptide chain length.
Separation: Proteins now migrate through the gel based solely on molecular weight.

The principle of SDS-PAGE (continued)

Gel Matrix: Polyacrylamide gel functions as a support for the electrophoresis.
Migration: High molecular weight proteins stay at the back, while low molecular weight proteins migrate faster.

Protein Identification

Albumin: Molecular weight: 66.5 kDa
Casein: Molecular weight: 24 kDa
Identification: Protein bands are observed during electrophoresis and can help determine the presence of proteins (and concentration) within a sample.

Materials/Equipment

Power Supplies
Gels: Prepared in the lab or precast gels
Vertical Gel Electrophoresis Chamber
Protein Samples
Running Buffer (Tris/Glycine/SDS)
Staining and Destaining Buffer
Protein Ladder (Prestained protein molecular weight standards)
Micropipettes and gel loading tips
Laemmli Sample Buffer: Used in sample preparation

Sample Preparation

Dilutions: Prepare dilutions of a 10% unknown protein solution (5%, 2.5%, and 1%) using water.
Sample Buffer: Add 1/4 the volume of Laemmli Sample Buffer to each protein solution.
Heat Treatment: Boil samples immediately after adding the Laemmli buffer to prevent protease activity from degrading proteins.

PROTOCOL

Dilutions: Prepare 1mL of 5%, 2.5%, and 1% solutions
Sample Addition: Add 15µL of each of the 3 dilutions to sample tubes.
Sample Buffer: Add 5μL of Laemmli buffer per sample tube
Gel Preparation:
- Add fresh running buffer to both chambers.
- Place protein ladder in the first well.
- Load samples.
Electrophoresis: Run gels at 180 volts for 30 minutes.
Visualisation: Stop the run when the dye front is near the bottom of the gel.
Staining and Destaining: Separate the gel plates, immerse in a staining solution (Coomassie Blue), and destain in deionized water; incubate with destaining solutions to remove excess stain.

Protein Gel Analysis

Single Band: Indicates a single protein type/subunit.
Multiple Bands: Indicates multiple protein types/subunits.
Relative Darkness (of Bands): Indicates protein concentration.
Protein Identity: Determine protein identity (e.g., Albumin or Casein) based on band patterns and molecular weight analysis.

Protein Ladder

Molecular Weight Markers: A mixture of proteins of known molecular weight used to identify the molecular weight of unknown proteins.
kDa: Kilodaltons, a unit for molecular size.

Question 1

Correct Answer: C. Smaller proteins migrate more rapidly through the gel

Question 2

Correct Answer: A. Staining them with the dye

Results

Gel Image: An image of the SDS-PAGE gel showing the different bands.
Protein Identification: Bands on the SDS-PAGE gel are used to identify proteins present in the sample.

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Description

Test your knowledge on key biochemistry lab techniques, specifically focusing on the use of Laemmli Sample Buffer and electrophoresis processes. This quiz covers important steps and precautions in protein sample preparation and analysis. Perfect for students in biochemistry or molecular biology courses!