MD100 Medical Biochemistry I: SDS PAGE Lab

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Questions and Answers

What is the primary purpose of boiling the samples at 70°C for 2 minutes?

To facilitate the transfer of proteins to the gel

To completely denature the proteins (correct)

To initiate the staining process

To enhance protein solubility

What is the role of Laemmli Sample Buffer in the preparation of protein samples?

To prevent protein degradation during the heating process

To provide a reducing environment and ensure protein denaturation (correct)

To maintain the protein's original structure

To modify the charge of the proteins

Which electrode in the electrophoretic apparatus should be connected to the bottom chamber?

Ground

Neutral

Cathode

Anode (correct)

What is the primary purpose of using sodium dodecyl sulfate (SDS) in the SDS-PAGE technique?

To denature proteins and make them negatively charged Signup and view all the answers

In SDS-PAGE, the migration of proteins occurs from which electrode to which electrode?

From cathode to anode Signup and view all the answers

How long should the gels typically run during the electrophoresis process?

30 minutes Signup and view all the answers

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

Urea Signup and view all the answers

What potential hazard is associated with 2-mercaptoethanol found in the Laemmli Sample Buffer?

It can cause respiratory distress if inhaled Signup and view all the answers

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

They tend to stay at the back of the gel Signup and view all the answers

What is the expected outcome if gels are overheated during electrophoresis?

Distortion of acrylamide or cracking of plates Signup and view all the answers

What step follows the addition of Coomassie Blue staining solution?

Rinsing with deionized water Signup and view all the answers

Which property of SDS allows it to effectively denature proteins in the sample?

Its anionic nature that imparts negative charge Signup and view all the answers

Which reducing agent is commonly used in conjunction with SDS for protein denaturation?

β-mercaptoethanol Signup and view all the answers

What is crucial to do before removing the gels after electrophoresis?

Turn off power and remove cables Signup and view all the answers

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

To serve as a reference for estimating protein sizes Signup and view all the answers

During gel analysis in SDS-PAGE, what is the expected location of albumin with a molecular weight of 66.5 kDa compared to casein with a molecular weight of 24 kDa?

Albumin remains at the back compared to casein 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

Introduction: SDS-PAGE laboratory technique
Theoretical Background: Principle of SDS-PAGE
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 molecular weight
Electrophoresis: Separation of macromolecules in an electric field
Support Medium: Discontinuous polyacrylamide gel
Denaturing Agent: Sodium dodecyl sulfate (SDS)
Principle: SDS binds to proteins, giving them a uniform negative charge, making protein migration dependent on size
Reducing Agent: β-mercaptoethanol cleaves disulfide bonds and helps to unfold proteins
Migration: Proteins migrate towards the anode (+ electrode)

The Principle of SDS-PAGE

Proteins: Initially folded with positive and negative charges
Reduction: Disulfide bonds are reduced by 2-mercaptoethanol
Linear Structure: Unfolded to a linear structure, with a negative charge proportional to the polypeptide chain length.

The Principle of SDS-PAGE (continued)

Migration: High molecular weight proteins migrate slower, while low molecular weight proteins migrate faster. Proteins migrate from the cathode (-) to the anode (+).

Protein Identification

Albumin: Molecular weight: 66.5 kDa
Casein: Molecular weight: 24 kDa

Materials/Equipment

Gels: Prepared in the lab or precast
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

Sample Preparation

Dilutions: Prepare 5%, 2.5%, and 1% dilutions from starting 10% unknown protein solutions.
Laemmli Sample Buffer: 1/4 volume of total sample volume, and include the following: Tris-HCI pH 6.8, SDS, Glycerol, 2-mercaptoethanol, Bromophenol Blue.
Heating: Heat protein samples immediately after adding Laemmli buffer to prevent protease degradation.

Protocol

Dilutions: Prepare dilutions of the unknown protein samples.
Sample Preparation: Combine protein sample with Laemmli buffer
Heat Samples: Completely denature the proteins for 2 minutes at 70°C
Add Buffer: Add running buffer to gel electrophoresis chambers.
Load Protein Ladder: Load 10µl of Protein Ladder at the first well
Load Samples: Load 10 µl per sample in the remaining wells.
Run Gels: Run gels at 180 volts.
Stop the Run: Stop when the dye front is near the bottom of the gel.
Gel Removal: Turn off power supply and disconnect cables.
Gel Stains: Soak the separated gel in Commassie Blue stain for 15 minutes on a rocking table.
Gel Destain: Rinse and destain the gel, adding fresh destain solution for additional 15 minute incubation and changing every 5 minutes (total 2 changes). Overnight incubation with de-ionized water.
Picture: Take pictures of the gels

Protein Gel Analysis

Bands: Single band implies only one protein, multiple bands imply multiple proteins or protein subunits
Identification: Use protein ladder to identify proteins.
Concentration Estimation: Relative band darkness implies relative protein concentration. Size comparisons will determine relative concentration.

Protein Ladder

Molecular weight markers: Used to determine the protein's approximate molecular weight.

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: A gel image showing multiple bands with molecular weight information.

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Description

This quiz explores the fundamentals of the SDS-PAGE laboratory technique covered in Medical Biochemistry I. Students will understand sample preparation, gel loading, staining, and analysis for protein identification. Perfect for those enrolled in the Fall 2024 semester at the European University Cyprus, School of Medicine.