biochemistry ppt

Biochemistry Part 1: SDS-PAGE

Introduction to SDS-PAGE

  • Definition: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used for the separation of proteins based on their size.

  • Mechanism: SDS-PAGE utilizes the method of electrophoresis to study proteins, separating them effectively by size.

Sample Preparation: Creating Lysates

  • Fundamentals: Before studying proteins, researchers must create a lysate from the cells or tissues of interest.

  • Process in Detail: Breaking open cells or tissues involves releasing cell content through homogenization, which can be achieved using various gentle mechanical techniques that preserve cellular components:
      1. Ultrasound: Utilizing high-frequency sound to break apart cells.
      2. Mild Detergents: Application of a mild detergent to form holes in the plasma membrane.
      3. High Pressure: Forcing cells through a small opening under high pressure.
      4. Shearing Forces: Using a close-fitting rotating plunger against thick walls of a glass vessel to shear cells.

  • Result: The result of homogenization is a thick mixture known as a homogenate or extract, comprising various cytosolic molecules such as:
      - Enzymes
      - Ribosomes
      - Metabolites
      - Membrane-enclosed organelles, largely intact.

Source of Lysates for the Experiment

  • Human Cancer Cell Lines: Specific lysates used in the study come from different human cancer cell lines:
      - Prostate Cancer: LNCaP and PC-3
      - Breast Cancer: MCF-7 and MDA-MB-231
      - Colon Cancer: HT-29 and HCT116.

Role of Sodium Dodecyl Sulfate (SDS)

  • Definition: SDS is a detergent that serves to solubilize proteins in the SDS-PAGE process.

  • Functionality: Individual polypeptide chains interact with SDS, forming negatively charged SDS-protein complexes.

  • Migration: During electrophoresis, these complexes migrate through polyacrylamide gels based on size.

Role of β-Mercaptoethanol (β-ME)

  • Definition: β-mercaptoethanol is a reducing agent utilized in the sample preparation for SDS-PAGE.

  • Functionality: It breaks disulfide linkages either within a single protein or between proteins:
      - This reduction allows the unfolded polypeptide chains to migrate effectively at a speed indicative of their molecular weight.
      - Typically, smaller proteins migrate more quickly through the gel than larger proteins.

Gel Electrophoresis Process

  • Electrophoresis: The technique that employs an electric field that prompts the migration of protein molecules in a solution. Migration speed and directional movement depend on:
      - Size of the protein
      - Net charge of the protein.
      - This properties form the foundation of the SDS-PAGE technique.

Experimental Procedure for SDS-PAGE

Setup

  1. Preparation: Begin by setting up the gel and gel assembly following the instructions provided in the BioRad manual. Ensure to remove the tape from the gel’s bottom.

  2. Running Buffer:
       - Prepare 1L of running buffer for each tank.
       - Add this buffer to both the inner and outer chambers of the gel tank.

  3. Sample Preparation:
       - Acquire the protein samples from the instructor.
       - Heat each sample for 5 minutes at 95°C prior to loading the gel.
       - Centrifuge the samples at top speed for 2 minutes to isolate the proteins.

  4. Loading the Gel:
       - Load the gel in the following order:
         - 10 μl of molecular weight (MW) marker
         - 20 μl of each lysate (20 μg) as detailed below:
           - Lane 1: MW marker
           - Lane 2: LNCaP
           - Lane 3: PC3
           - Lane 4: MCF-7
           - Lane 5: MDA-MB-231
           - Lane 6: HT-29
           - Lane 7: HCT-116
           - Lane 8: 20 μl of 1X sample buffer to ensure the samples run straight.

  5. Running the Gels:
       - Run a total of 3 gels per tank at 200 volts until the dye front reaches the bottom of the gel.

Post-Electrophoresis

  1. Preparing for Protein Transfer:
       - Simultaneously prepare the PVDF membrane and ion reservoir stacks as follows:
         - PVDF Membrane:
           - Soak the PVDF membrane in 100% methanol until translucent.
           - Equilibrate the membrane in 1X transfer buffer for 2-3 minutes.
         - Transfer Stacks:
           - Immerse two stacks in soaking trays with 50 ml of 1X transfer buffer for 2-3 minutes each.

  2. Removing the Gel:
       - After electrophoresis, turn off the power and disconnect leads.
       - Remove the gel by pouring off and discarding the running buffer and carefully open the cassette to take the gel out.

  3. Transferring Proteins to PVDF Membrane:
       - Conduct protein transfer onto the PVDF membrane using the designated Turbo protocol outlined in the Turbo Transfer video.

Post Transfer: Membrane Storage Protocol

  • Storage of Blots: Post-transfer, retaining the integrity of the PVDF membrane for future analysis is critical:
      1. Place the dry PVDF blot between two clean Whatman 3MM paper sheets.
      2. Sandwich the blot between two sheets of card stock or thin cardboard.
      3. Use paper clips to secure the edges together.
      4. Seal the sandwich in a plastic bag.
      5. Storage options:
         - 4˚C: Up to 2 weeks
         - -20˚C: Up to 2 months
         - -70˚C: For longer-term storage.

Notes and Administrative Details

  • Reminders: Students are required to submit their carbon copy notebooks.

  • Group Postlab: A group postlab is available on Sakai, which is due at the beginning of the next lab session.