MD100 Biochemistry I - Lab Exercise 3 SDS-PAGE

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Questions and Answers

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

To enhance the visibility of proteins

To prevent the degradation of denatured proteins (correct)

To activate proteases for protein digestion

To increase the protein concentration

How much Laemmli Sample Buffer should be added to each protein sample?

10μl

25μl

5μl (correct)

15μl

What is the correct order of steps after adding the protein samples to the gel?

Load the ladder, run the gel, load samples

Load samples, run the gel, load the ladder

Run the gel, load samples, load the ladder

Load the ladder, load samples, run the gel (correct)

Why is it important to connect the anode and cathode correctly while setting up the electrophoretic apparatus?

To ensure that negatively charged proteins migrate toward the anode Signup and view all the answers

What is the primary purpose of SDS-PAGE in protein analysis?

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

What is the correct voltage setting for running the gels, according to empirical methods?

180 volts Signup and view all the answers

In SDS-PAGE, what is the role of sodium dodecyl sulfate (SDS)?

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

What should be done right before removing the gels after electrophoresis?

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

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

They stay closer to the starting point of the gel. Signup and view all the answers

What is the role of Coomassie Blue in the protein gel procedure?

To stain the proteins for visualization Signup and view all the answers

Which buffer is commonly used in SDS-PAGE for running the gel?

Tris/Glycine/SDS Signup and view all the answers

During the destaining process of the gel, how often should the destain solution be changed?

Every 5 minutes Signup and view all the answers

What is the function of reducing agents like β-mercaptoethanol in SDS-PAGE?

To cleave disulphide bonds in proteins. Signup and view all the answers

Which component is NOT part of the Laemmli Sample Buffer used for protein denaturation?

EDTA Signup and view all the answers

Why is a protein ladder used in SDS-PAGE?

To allow for molecular weight estimation of unknown proteins. Signup and view all the answers

What is the direction of protein migration in SDS-PAGE?

From cathode (-) to anode (+) Signup and view all the answers

Study Notes

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

Course: MD100 Medical Biochemistry I
Lab Exercise: 3 - Introduction to SDS PAGE - Protein identification and characterization
Semester: Fall 2024
Institution: European University Cyprus, School of Medicine

Objectives

SDS-PAGE: Introduction to the 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
SDS-PAGE Support: Uses discontinuous polyacrylamide gel as the support medium
SDS (Sodium Dodecyl Sulfate): Denatures proteins and gives them a uniform negative charge.
Principle: Charged molecules migrate towards the oppositely charged electrode in an electric field. SDS binds to proteins to make them uniformly negatively charged. When a current is applied, all SDS-bound proteins migrate towards the positive electrode
Reducing Agents (beta-mercaptoethanol): Cleave disulfide bonds (S-S) and disrupt protein structure along with SDS

The principle of SDS-PAGE

Protein Structure: Proteins start folded with positive and negative charges
Reduction with Beta-mercaptoethanol: Disulfide bonds (S-S) are cleaved, rendering linear chain structure.
SDS Binding: Each polypeptide chain is surrounded by a negative charge of SDS proportional to its length
Migration: Proteins move in the gel according to the molecular weight (smaller proteins migrate faster); (larger proteins migrate more slowly)

The principle of SDS-PAGE - (Electropherogram)

Migration Direction: Proteins migrate from the cathode (-) to the anode (+)
Molecular Weight: Larger proteins migrate slower and are located at the top. Smaller proteins migrate faster and located at the bottom
SDS-PAGE support (Gel): This is polyacrylamide gel

Protein identification

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

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

Sample Preparation

Dilutions: 5%, 2.5%, and 1% dilutions from 10% unknown protein solution
Sample Buffer Addition: 4x stock Laemmli sample buffer in a 1/4 dilution ratio to the sample volume

Sample Preparation (cont.)

Laemmli Sample Buffer Preparation:
- Tris-HCl pH 6.8
- SDS
- Glycerol
- 2-mercaptoethanol
- Bromophenol Blue
Heating Samples: Important to heat protein samples immediately after adding Laemmli buffer to prevent protease degradation of Denatured proteins

PROTOCOL

Dilution Preparation: Prepare 1mL of 5%, 2.5%, and 1% dilutions from a 10% unknown protein solution.
Sample Addition: Add 15 µL of 5%, 2.5%, and 1% unknown protein samples into a new tube.
Sample Buffer Addition: Add 5 µL of 4x stock Laemmli Sample Buffer to each sample (1/4 of total sample volume).
Boiling Samples: Boil samples to denature proteins at 70°C for 2 minutes in a heat block
Running Buffer Addition: Add freshly prepared 1X Running Buffer to the gel chambers
Protein Ladder: Load 10 µL of a Protein Ladder in the first well of the gel
Sample Loading: Load 10µL of samples to the gel
Gel Run: Run the gels at 180 volts for approximately 30 minutes (stop when dye front is near gel bottom)
Gel Staining: Separate the gel plates, drop the gel into a staining dish containing deionized water and add Coomassie Blue staining solution for 15 minutes on a rocking table. (Ensure Coomassie Blue stain covers gel)
Destaining: Pour off stain and rinse with deionized water. Add fresh destain, incubate for 15 minutes with every 5-minute change; repeat 2 times (total of 2 changes), add deionized water for overnight incubation on a rocking table.
Photograph: One representative from each group should gather data, including a photograph of the gel analysis

Protein Gel Analysis

Single Protein/Subunit: Protein samples containing only one protein type will have one band in the image.
Multiple Proteins/Subunits: Multiple bands indicate the presence of multiple proteins or subunits.
Albumin/Casein: You can identify which bands correspond to albumin or casein based on the results.
Protein Concentration: Estimate protein concentration by comparing the band intensity to that of a known amount of each protein or standard protein ladder.

Protein Ladder (Molecular Weight Markers)

Molecular Weight Ranges: Provide a known molecular weight scale on the gel, allowing for estimates of the molecular weights of unknown proteins. (kDa values shown on the ladder image)

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: Shows the results for identification of unknown proteins.
kDa Values: The gel image has an associated molecular weight (kDa) reference scale for identification.

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Description

This quiz covers Lab Exercise 3 of MD100 Medical Biochemistry I, focusing on SDS-PAGE. Learn about protein identification and characterization through sample preparation, gel loading, staining, and analysis techniques. Perfect for students aiming to grasp the fundamentals of electrophoresis in protein research.