SDS-PAGE Techniques and Protocols

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Questions and Answers

What is the purpose of boiling the protein samples at 70°C after adding Laemmli Sample Buffer?

To promote the protein crystallization process

To activate the proteases for better protein manipulation

To denature the proteins and prevent protease activity (correct)

To enhance the protein's tertiary structure

What should the proportion of Laemmli Sample Buffer be in relation to the total sample volume?

Equal to the total sample volume

1/4 of the total sample volume (correct)

1/2 of the total sample volume

1/3 of the total sample volume

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

To stain the proteins during electrophoresis

To stabilize the protein samples during storage

To break up the protein structure and add negative charge (correct)

To enhance the visibility of the protein bands

Why must the power be turned off before removing the gels from the electrophoretic apparatus?

To ensure safety while handling electrical equipment Signup and view all the answers

What is the ideal voltage used for running the gels during electrophoresis?

180 volts Signup and view all the answers

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

Every 5 minutes Signup and view all the answers

What precaution should be taken when using 2-mercaptoethanol in Laemmli Sample Buffer?

It is toxic if inhaled or absorbed through the skin Signup and view all the answers

What is the first step after electrophoresis is completed?

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

What is the primary purpose of using SDS in SDS-PAGE?

To denature proteins and impart a negative charge Signup and view all the answers

Which of the following statements about the migration of proteins during SDS-PAGE is true?

Proteins are separated primarily based on their molecular weight Signup and view all the answers

What role do reducing agents play in the SDS-PAGE process?

They cleave disulfide bonds within proteins Signup and view all the answers

What is the significance of using a protein ladder during SDS-PAGE?

To estimate the molecular weight of separated proteins Signup and view all the answers

What type of gel is used in the SDS-PAGE technique?

Discontinuous polyacrylamide gel Signup and view all the answers

Which of the following components is NOT part of the Laemmli Sample Buffer?

Sodium Phosphate Signup and view all the answers

Why do low molecular weight proteins migrate faster during SDS-PAGE compared to high molecular weight proteins?

They encounter less friction in the gel matrix Signup and view all the answers

What does the term 'electrophoresis' refer to in the context of SDS-PAGE?

The movement of charged molecules in an electric field 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: Introduction to the laboratory technique
- Theoretical Background: Principle of SDS-PAGE
- Part A: Sample preparation & dilutions
- Part B: Sample loading & gel running
- Part C: Gel staining & 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-PAGE Medium: Uses discontinuous polyacrylamide gel as the support medium. Sodium dodecyl sulfate (SDS) is used to denature the proteins.
SDS Principle: A negatively charged molecule migrates to the electrode within the electric field with an opposite polarity. SDS denatures and binds to proteins, resulting in uniform negative charging to ensure all components in the sample will move through the gel towards the positive electrical pole.
Reducing Agents: β-mercaptoethanol is used to reduce disulfide bonds within the proteins, further causing denaturation alongside SDS.

The Principle of SDS-PAGE

Protein Structure: Before SDS-denaturation, proteins exist in a folded 3D structure from positive and negative charged amino acids.
Disulfide Bonds: Proteins usually have disulfide bonds connecting certain amino acids. SDS alongside a reducing agent (like β-mercaptoethanol), unfolds the linear polypeptide chain while breaking the bonds.
SDS Binding: The denatured linear structure is then bound by anionic detergent SDS, creating a strong negative charge proportional to the polypeptide chain length.
Migration: When an electric field is applied, all SDS-bound proteins migrate through the gel towards the positively charged electrode, separating proteins based on the size and length of their polypeptide chain (molecular weight).

Protein Identification

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

Materials & Equipment

Gels: Prepared in lab or precast gels purchased from the market
Electrophoresis Chamber: Vertical Gel Electrophoresis Chamber
Protein Samples: Samples of interest to be studied.
Buffers: Running Buffer (Tris/Glycine/SDS), Staining, & Destaining Buffer
Protein Ladder: Prestained protein molecular weight standards
Tools/Supplies: Micropipettes, gel loading tips
Laemmli Sample Buffer

Sample Preparation

Dilutions: The provided concentration of protein sample (10%) is diluted to 5%, 2.5%, and 1%.
Laemmli Sample Buffer: Laemmli Sample Buffer is added to each sample. Equal volumes are added for each concentration.
Heating Proteins: Heating proteins with Laemmli Sample Buffer immediately after adding it is important to prevent protein degradation from proteases, which may be resistant to SDS.

Protocol

Dilutions: Prepare 1ml dilutions (5%, 2.5%, and 1%) of the protein sample (10%) by volume.
Adding Samples: Add 15µl of each dilution to the separate tubes.
Labeling Tubes: Properly label all tubes to be used.
Sample Buffer Addition: Added 5µl of 4x stock Laemmli sample buffer to each tube.
Heating: Boil samples in a water bath (70°C) for 2 minutes. This exactly denatures the protein.
Running Conditions: Run gels at 180 volts for approximately 30 minutes. Stop the run once the dye front reaches the bottom of the gel.
Gel Staining: Separate gel plates carefully, and add the gel to the staining dish with diluted deionized water, then add Coomassie Blue Stain solution for 15 minutes.
Destaining: Remove the solution, add deionized water, leave overnight to destain. Add fresh solution, run for 15 minutes, and change solution every 5 minutes twice in total then change to water.
Picture: One student from each group takes pictures of the gel the following morning.

Protein Gel Analysis

Lanes with one band: Indicate the sample containing only one protein or protein subunit
Multiple Bands: Indicate more than one protein or protein subunits/polypeptides.
Identification: Estimate the identity of the proteins in each lane (identification is not mentioned in the protocol).
Protein Concentration: The band's darkness indicates the protein concentration in the solution. The band size reflects the relative concentration.

Protein Ladder

kDa: Molecular weights marked on the protein ladder include: ~180, ~130, ~100, ~75, ~63, ~48, ~35, ~28, ~17, ~10
Gel: A tris-glycine 4-20% polyacrylamide gel

Question 1

Correct Answer: Smaller proteins migrate more rapidly through the gel in an SDS-PAGE process. (C)

Question 2

Correct Answer: Proteins are visualized by staining with a dye. (A)

Results

Image Description: This section includes an image of the completed gel electrophoresis results. The image provides a visual representation of the separated proteins. The labels and sizes are included in this image.

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Description

This quiz covers essential techniques and protocols related to SDS-PAGE, including the proper use of Laemmli Sample Buffer and the role of SDS. Test your knowledge on sample preparation, electrophoresis procedures, and the significance of using a protein ladder. Perfect for students and researchers in biochemistry and molecular biology.