SDS-PAGE Techniques and Protocols

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.