Laboratory Techniques in Bioinformatics: SDS-PAGE and RFLP Analysis

Learning Outcomes

  • Understand the principles and applications of SDS-PAGE analysis.

  • Gain knowledge in recombinant protein expression.

  • Analyze Restriction Fragment Length Polymorphisms (RFLP).

  • Develop competency in setting up protocols for genomic DNA extraction and PCR reactions.

Key Points on SDS-PAGE Analysis

  • Definition: SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a method used to visualize and examine proteins.

  • Applications: Analyzing changes in protein concentration, examining recombinant proteins, assessing protein changes related to diseases (e.g., red blood cell membranes).

Preparation for Electrophoresis

  • Sample Preparation: Treat cells/bacteria with detergent to lyse cells.

  • Sample Buffer Components:

    • SDS: Provides a negative charge.

    • β-mercaptoethanol: Reduces disulfide bonds.

    • Other components: Buffer, salt, bromophenol blue (tracking dye), glycerol (to increase density).

  • Boiling Proteins: Proteins must be boiled at 95°C for 5 minutes to denature them for size separation.

Mechanism of SDS

  • SDS binds to proteins, denaturing them and giving them a uniform negative charge for separation based on size.

Gel Composition for SDS-PAGE

  • Acrylamide Gels separate proteins by size:

    • 15% gel: 12-43 kDa

    • 10% gel: 16-68 kDa

    • 7.5% gel: 36-94 kDa

    • 5.0% gel: 57-212 kDa

Gel Loading

  • Load samples into gel wells:

    • Molecular weight marker: 5 µL

    • GST protein: 10 µL

    • Lysates (blue and white): 10 µL each.

  • Electrophoresis Conditions: Run at approximately 150V for about one hour.

Protein Visualization

  • Staining is required after gel run with Coomassie blue to observe proteins.

  • Electrophoresis Mechanism: Smaller proteins migrate faster, larger proteins migrate slower.

RFLP Analysis

  • RFLP detects single nucleotide changes (SNPs) in DNA sequences.

    • Example: Detection of SNP mutation using EcoR1:

    • Normal Sequence: 5’ GAATTC 3’

    • Mutated Sequence: 5’ GAAGTC 3’

  • Sequencing examples from hemoglobin:

    • Normal Hb: ACTCCTGAGGAG

    • Sickle Cell Hb: ACTCCTGTGGAG

  • BseR1 Digestion: Differentiates wild-type from mutated sequences after PCR amplification.

Safety Measures in Laboratory

  • Wear full PPE: lab coats, gloves, and eye protection.

  • Handle cheek swabs with caution due to potential COVID risks, and report spillages immediately.

Conclusion

  • Clean workspace and dispose of waste appropriately after experiments, using Chemgene to clean surfaces effectively.