biochemistry

Biochemistry Week One - SDS-PAGE Study Notes

Preparation Before Lab

  • Reading and Videos
      - Read the entire lab document.
      - Watch instructional videos on:
        - Mini-PROTEAN® TGX™ Precast Gels.
        - Trans-Blot® Turbo™ Transfer System.

  • Purpose Statement
      - Write a clear, concise purpose statement in your lab notebook.

During Lab

  • Documentation
      - Write down the procedure step-by-step.
      - Note any changes to the protocol or mistakes made during the lab.

Introduction to SDS-PAGE

  • Overview of SDS-PAGE
      - Definition: SDS-PAGE stands for sodium dodecyl sulfate - polyacrylamide gel electrophoresis, a method for resolving proteins in complex mixtures.
      - Objective: Use SDS-PAGE to separate proteins extracted from various human cancer cell lines:
        - Prostate cancer cell lines: LNCaP and PC-3.
        - Breast cancer cell lines: MCF-7 and MDA-MB-231.
        - Colon cancer cell lines: HT-29 and HCT116.
      - After separation, proteins will be transferred to a membrane (PVDF) for analysis.
      - Next Steps: In Week Two, introduction to immunoblotting (Western blotting) with a focus on epithelial to mesenchymal transition (EMT).

What is EMT?

  • Definition: A biological process where epithelial cells lose their characteristics and acquire mesenchymal features.

  • Characteristics of EMT:
      - Loss of cell-cell junctions.
      - Loss of apical-basal polarity.
      - Loss of epithelial markers.
      - Gain of cell motility and spindle-shaped morphology.
      - Acquisition of mesenchymal markers.

  • Significance: Associated with increased tumor aggressiveness and migration from the primary site.

Background Information

  • SDS-PAGE Technique
      - Enables analysis of proteins in complex extracts through a discontinuous system derived from Laemmli (1970).
      - Components:
        - Stacking Gel: Concentrates proteins before entering the resolving gel.
        - Resolving Gel: Separates proteins based on molecular weights.

  • Separation Process:
      - Proteins migrate through the gel, with smaller proteins moving faster due to less resistance.
      - Factors influencing migration include size, structure, and charge of the proteins.

Role of SDS in SDS-PAGE

  • Sodium Dodecyl Sulfate (SDS)
      - Function: Denatures proteins and binds to the protein backbone at a constant molar ratio.
      - Impact on Proteins:
        - Unfolds into linear chains with an overall negative charge proportional to their length.
        - Eliminates the influence of protein structure and charge, allowing separation solely by length.

  • Polyacrylamide Gel Development:
      - Forms a mesh-like matrix for size-based protein separation.
      - The size of pores can be adjusted by changing acrylamide concentrations.

Overview of SDS-PAGE Procedure

  • Sample Preparation
      - Isolation: Proteins are isolated from cells/tissues via lysis.
      - Lysis Process: Breaks down the cell membrane to separate soluble proteins.
      - Lysis Buffers: Vary in detergent strength, ensuring soluble proteins are released.
      - Sample Buffer (2x Laemmli sample buffer):
        - Contains glycerol (to sink samples) and bromophenol blue (tracking dye).
        - SDS and a reducing agent included for complete denaturation.
        - Samples are boiled to aid denaturation.

Electrophoresis and Blotting

  • Gel Electrophoresis:
      - Denatured samples, uniformly negatively charged due to SDS, migrate in an electric field towards the positive electrode.
      - Separation based on size primarily, with the charge-to-mass ratio equalized by SDS binding.

  • Transfer Process:
      - After separation, proteins are transferred onto a solid support (PVDF membrane) for analysis.
      - Electroblotting (Towbin et al., 1979) is preferred for its speed and efficiency, using an electric field to move proteins out of the gel onto the membrane.

  • Membrane Placement:
      - Membrane goes between the gel and the positive electrode for proper migration.

Types of Blotting Membranes

  • Nitrocellulose Membrane:
      - Pros: Excellent protein binding and retention capabilities.
      - Cons: Brittle, less effective for reuse.

  • Polyvinylidene Fluoride (PVDF) Membrane:
      - Pros: Superior mechanical strength, good for stripping/reprobing and further protein characterization techniques (sequencing, proteolysis).
      - Cons: Higher background staining, requires careful handling.

References

  1. Laemmli UK. "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature, 1970 Aug 15; 227(5259):680-5. doi: 10.1038/227680a0. PMID: 5432063.

  2. Towbin H, Staehelin T, Gordon J. "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications." Proc Natl Acad Sci U S A, 1979 Sep; 76(9):4350-4. doi: 10.1073/pnas.76.9.4350. PMID: 388439; PMCID: PMC411572.

Experimental Protocol: Running SDS-PAGE

  1. Set up the gel and gel assembly according to the BioRad manual:
      - Ensure tape from the bottom of the gel is removed.

  2. Prepare 1L of running buffer per tank; add running buffer to inner and outer chambers.

  3. Obtain samples from the instructor.

  4. Samples should be heated for 5 minutes at 95°C before loading.

  5. Centrifuge samples at top speed for 2 minutes if necessary.

  6. Loading Gel:
      - Load the following:
        - 10 µl of the molecular weight (MW) marker.
        - 20 µl of each lysate (20 µg) in the designated order:
          - Lane Order: 1 MW marker, 2 LNCaP, 3 PC-3, 4 MCF-7, 5 MDA-MB-231, 6 HT-29, 7 HCT-116, 8 1x sample buffer (20 µl).

  7. Run gels at 200 volts until the dye front reaches the bottom of the gel.

  8. Prepare PVDF Membrane and Ion Reservoir Stacks:
      - Write table number on lower left corner of the membrane.
      - Wet the PVDF membrane in MeOH:
        - Immerse in 100% methanol until translucent.
        - Soak in 1x transfer buffer for 2-3 minutes.
        - Wet transfer stacks by immersing in 50 ml of 1x transfer buffer for 2-3 minutes.

  9. Disassemble the gel and set up the transfer:
      - Place a wetted transfer stack on the bottom of the cassette, removing air bubbles.
      - Place the wetted PVDF membrane on top of the stack in the cassette, removing air bubbles.
      - Place the gel on the PVDF membrane with MW markers on the left, rolling out air bubbles.
      - Top with another wetted transfer stack, removing air bubbles.

  10. Remove excess transfer buffer by inverting the cassette base; lock the cassette lid.

  11. Select Turbo protocol on the Trans-Blot® Turbo™ for protein transfer.

  12. After transfer, rinse membranes with dH2O and place on Whatman paper to dry.

  13. Visualize proteins by UV activating the membrane.