MD100 Medical Biochemistry I

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Questions and Answers

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

To stabilize the proteins before electrophoresis

To denature the proteins effectively (correct)

To add a positive charge to the amino acids

To increase the viscosity of the samples

Why must the samples be boiled after adding Laemmli buffer?

To neutralize the acidity of the buffer

To completely denature the proteins (correct)

To promote the polymerization of acrylamide

To enhance the binding of the Coomassie Blue dye

At what voltage should the gels be run as specified in the protocol?

180 volts (correct)

150 volts

200 volts

120 volts

How long should the gels be run during electrophoresis?

30 minutes Signup and view all the answers

What must be done before removing the gels from the electrophoresis apparatus?

Disconnect the power supply and remove cables Signup and view all the answers

Which component in the Laemmli buffer is toxic and should be handled carefully?

2-mercaptoethanol Signup and view all the answers

What is the primary role of SDS in the sample preparation process?

To break down protein structures and add negative charge Signup and view all the answers

What is the purpose of the incubation with Destain solution?

To remove excess Coomassie Blue stain Signup and view all the answers

What is the main purpose of SDS in the SDS-PAGE technique?

To denature proteins and ensure they carry a uniform negative charge Signup and view all the answers

In the SDS-PAGE process, which factor primarily influences the rate of migration of proteins through the gel?

The molecular weight of the proteins Signup and view all the answers

Which of the following components is NOT typically included in the Laemmli Sample Buffer?

Sodium chloride Signup and view all the answers

What is the function of reducing agents such as beta-mercaptoethanol in the SDS-PAGE procedure?

To cleave disulfide bonds and disrupt protein structure Signup and view all the answers

In an SDS-PAGE gel, where do high molecular weight proteins typically end up after running the gel?

At the back of the gel Signup and view all the answers

What is the role of the protein ladder in SDS-PAGE?

To serve as a molecular weight standard for protein identification Signup and view all the answers

During the sample loading phase of SDS-PAGE, what is the significance of having bromophenol blue in the Laemmli Sample Buffer?

It provides a colorimetric indicator for sample loading and migration Signup and view all the answers

What is the result of the discontinuous nature of the polyacrylamide gel in SDS-PAGE?

Enhances the resolution of separating proteins based on size 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
Objectives:
- Introduction to SDS-PAGE laboratory technique
- Theoretical background: Principle of SDS-PAGE
- Sample preparation: Dilutions
- Sample loading: Running the gel
- Gel staining and destaining
- Protein gel analysis: Protein identification

SDS-PAGE Principle

Technique: SDS-PAGE separates proteins based on molecular weight.
Electrophoresis: The separation of macromolecules in an electric field.
SDS-PAGE Medium: Uses polyacrylamide gel as a support medium.
SDS (Sodium Dodecyl Sulfate): A detergent that denatures proteins and gives them a uniform negative charge.
Principle: Charged molecules in an electric field migrate towards the oppositely charged electrode.
Protein Denaturation: SDS disrupts protein structure, and reducing agents like β-mercaptoethanol break disulfide bonds.
Migration: The proteins move through the gel from cathode to anode based on size. Smaller proteins migrate faster.

Protein Identification

Gel Analysis: Analyzing protein bands (colors/darkness) to determine protein identity and concentration.
Molecular Weight Markers: Known proteins/molecules of known molecular weight used to determine the molecular weight of the unknown protein samples.

Materials and Equipment

Power supplies
Gels (prepared in lab or precast)
Vertical Gel Electrophoresis Chamber
Protein Samples
Running Buffer (Tris/Glycine/SDS)
Staining and Destaining Buffer
Protein Ladder (Prestained protein MW standards)
Micropipettes and gel loading tips
Laemmli Sample Buffer

Sample Preparation

Dilutions: Preparing 5%, 2.5%, and 1% dilutions of a 10% unknown protein solution.
Laemmli Sample Buffer: Includes Tris-HCl (pH 6.8), SDS, glycerol, 2-mercaptoethanol, and bromophenol blue.
Heat Treatment: Heat protein samples in Laemmli buffer to prevent degradation by protease enzymes. SDS must be added to the samples, before denaturation step.

Protocol

Step 1:* Prepare protein dilutions.
Step 2:* Add 15µL of diluted protein to new tubes.
Step 3:* Add 5µL of Laemmli buffer to each sample(1/4 of total sample volume).
Step 4:* Boil protein samples to denature them at 70°C for 2 minutes.
Step 5:* Add 1x Running buffer to gel chambers.
Step 6:* Load 10µL of protein ladder in the first lane of the gel.
Step 7:* Load 10µL of each sample to the gel.
Step 8:* Run gels at 180 volts for 30 minutes or until the dye reaches the bottom.
Step 9:* Turn off power, remove cables
Step 10* Separate gel plates, and transfer gel to deionized water.
Step 11:* Transfer and add coomassie blue stain the solution (15 minutes on a rocking table).
Step 12:* Pour off stain solution and rinse the gel in deionized water.
Step 13:* Add fresh Destain solution to cover the gel(incubation of 15 minutes and change every 5 minutes).
Step 14:* Remove Destain and add deionized water for overnight in a rocking table.
Step 15:* Take photographs of gels.

Protein Gel Analysis

Protein Bands: Single bands show one protein, multiple bands demonstrate multiple proteins.
Protein Identification: Determining the identity by comparing the size to known samples(protein ladder).

Questions

Question 1:* In SDS-PAGE, what happens regarding proteins?
Answer: Proteins are denatured and have a uniform negative charge. Smaller proteins migrate faster.
Correct option: Proteins are denatured by SDS
Question 2:* How are proteins visualized on gels?
Answer: By staining with a dye.
Correct answer: Staining them with the dye.

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Description

This quiz focuses on the SDS-PAGE laboratory technique used for protein identification and characterization in Medical Biochemistry I. It covers the principles of SDS-PAGE, sample preparation, and analysis methods. Enhance your understanding of protein separation based on molecular weight through this comprehensive exercise.