Medical Biochemistry I - Lab Exercise 3: SDS-PAGE

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).

• Parts: Breakdown of the lab exercise into parts: sample preparation (including dilutions), sample loading and running gel, gel staining and destaining, protein gel analysis (protein identification).

Introduction to SDS-PAGE

• Technique: Used to separate proteins based on their molecular weight through electrophoresis.

• Electrophoresis: The separation of macromolecules in an electric field is called electrophoresis.

• SDS-PAGE: In SDS-PAGE, a polyacrylamide gel is the support medium and sodium dodecyl sulfate (SDS) is used to denature the proteins.

• Principle: A charged molecule migrates to the oppositely charged electrode when placed in an electric field. SDS, working as an anionic detergent, denatures and binds to proteins, uniformly giving them a negative charge. This ensures all SDS-bound proteins migrate in the same direction (toward the anode) when current is applied, regardless of their initial charge.

• SDS effect: Reducing agents (e.g., β-mercaptoethanol) cleave disulphide bonds and disrupt protein structure.

The principle of SDS-PAGE (detailed)

• Protein structure: Proteins initially have complex folded structures with positive and negative charges.

• Reduction: Reducing agents like 2-mercaptoethanol (2-ME) break the disulfide bonds in proteins and cause them to unwind.

• SDS binding: SDS molecules bind to the unfolded proteins and impart a uniform negative charge, which is proportional to the length of the polypeptide chain. This effectively negates any initial positive and negative charges of the proteins.

• Migration: When the protein mixture is subjected to an electric field, the negatively charged protein-SDS complexes will migrate towards the anode (positive electrode), with smaller proteins travelling faster than larger ones due to less obstruction within the polyacrylamide gel matrix.

• Molecular weight separation: This allows separation of proteins based on their molecular weight—smaller proteins migrate a greater distance along the gel.

Protein Identification

• Albumin: Molecular weight: 66.5 kDa.

• Casein: Molecular weight: 24 kDa.

Materials/Equipment

• Gels: Prepared in lab or pre-cast.

• Electrophoresis Chamber: Vertical gel electrophoresis chamber.

• Protein Samples: Sample solutions for analysis.

• Buffers: Running buffer (Tris/Glycine/SDS), staining/destaining buffers.

• Protein Ladder: Pre-stained protein molecular weight standards.

• Micropipettes/tips: For accurate sample/buffer delivery.

• Laemmli Sample Buffer: Used for sample preparation, containing components like Tris-HCl buffer, SDS, glycerol, 2-mercaptoethanol, and bromophenol blue.

Sample Preparation

• Dilutions: Create 5%, 2.5%, and 1% dilutions of 10% unknown protein sample.

• Laemmli Buffer Addition: Add 5 µL of 4x Laemmli stock buffer to each dilution. The important note here is that the Laemmli sample buffer should be ¼ of the sample volume.

• Heating for Protein Denaturation: Heat the samples at 70°C in a heat block for 2 min to disrupt the protein structure. SDS enhances this denaturing function and renders the resulting proteins uniformly negatively charged

Protocol

• Dilutions: Prepare dilutions of unknown protein solution with water.

• Sample Addition: Add precise volumes of each dilution to new labeled tubes.

• Laemmli Addition: Add Laemmli Sample Buffer to each tube.

• Heat Denaturation: Boil the mixed samples to denature proteins in a heat block at 70°C for 2 min.

• Buffer Addition: Add running buffer to both chambers of the electrophoresis apparatus.

• Ladder Loading: Load the protein ladder in the first lane of the gel.

• Sample Loading: Load other protein samples in the following lanes.

• Electrophoresis: Run the gel at 180 volts for roughly 30 min, until the dye front reaches the bottom of the gel.

• Gel Staining: Remove the separated proteins from the gel and transfer it to a staining dish filled with deionized water and Coomassie Blue staining solution for 15 minutes with rocking.

• Destaining: Remove the Coomassie blue staining solution, rinse with deionized water multiple times.

• Overnight Destain:* Remove the staining solution and fill the staining dish with deionized water, incubating the gel overnight while rocking to ensure complete removal.

• Destain Changes: Perform two washes, each about 15 minutes, for total 30 minutes of destaining. Change the destaining solution with fresh solution every 5 mins.

• Gel Picture: Take a picture of the gel to analyse.

Protein gel analysis

• Band interpretation: Bands in the gel indicate the presence of proteins or protein subunits. Single bands for a lane suggest one protein/protein subunit; multiple bands mean multiple proteins or subunits/polypeptides.

• Protein identification: Determine protein identity by comparing the migration distances of unknown sample bands to the bands from known proteins (like the protein ladder).

• Protein concentration: Assess protein concentration by the intensity (darkness) of the bands.

Additional

• Question 1: The correct answer is C. Smaller proteins migrate faster due to less obstruction through the gel than larger ones

• Question 2: The correct answer is A. Proteins are visualized using dye staining on gels

• Results: Sample gel image illustrating the identified protein bands.

• Protein Ladder: The protein ladder is used to gauge the molecular weight of unknown proteins in the samples.