MD100 Medical Biochemistry I - Lab Exercise 3

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.