Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide Gel Electrophoresis (PAGE) is a widely used technique for separating proteins based on their size and charge. 

Clinical Significance

1. Protein Analysis: PAGE is used to analyze protein mixtures, including detecting abnormal protein patterns in diseases such as multiple myeloma, autoimmune disorders, and certain cancers.

2. Genetic Research: Identifies genetic variations and mutations by analyzing proteins or nucleic acids.

3. Diagnostics: Helps in diagnosing conditions by analyzing serum proteins, hemoglobin variants, and enzyme deficiencies.

4. Biomarker Discovery: Essential in the discovery and validation of biomarkers for various diseases.

Principle

PAGE separates proteins based on their electrophoretic mobility, which is a function of the length, conformation, and charge of the molecule. The gel matrix acts as a sieve, with smaller molecules moving faster than larger ones.

Requirements

- Equipment: 

  - PAGE apparatus (gel casting unit, electrophoresis tank)

  - Power supply

  - Gel documentation system

- Reagents:

  - Acrylamide and bis-acrylamide

  - Ammonium persulfate (APS)

  - TEMED (N,N,N',N'-tetramethylethylenediamine)

  - Tris buffer

  - SDS (sodium dodecyl sulfate, for SDS-PAGE)

  - Loading buffer (with dye, e.g., bromophenol blue)

  - Running buffer (e.g., Tris-glycine)

  - Protein standards/markers

  - Staining solutions (Coomassie Brilliant Blue, Silver Stain)

Procedure

1. Gel Preparation:

   - Mix acrylamide, bis-acrylamide, Tris buffer, APS, and TEMED to prepare the gel solution.

   - Pour the solution into a casting mold with a comb to create wells.

   - Allow the gel to polymerize.

2. Sample Preparation:

   - Mix protein samples with loading buffer.

   - Heat the mixture to denature proteins (for SDS-PAGE).

3. Loading and Running the Gel:

   - Place the gel in the electrophoresis tank and add running buffer.

   - Load the protein samples and molecular weight markers into the wells.

   - Connect the apparatus to a power supply and run the electrophoresis at a constant voltage.

4. Staining and Destaining:

   - After electrophoresis, immerse the gel in staining solution to visualize the proteins.

   - Destain the gel to remove excess stain, allowing clear visualization of the protein bands.

Interpretation

- Band Pattern: Compare the pattern of bands in the sample lanes to the molecular weight markers to determine the size of the proteins.

- Band Intensity: The intensity of the bands correlates with the quantity of the protein.

- Mobility: The relative mobility of the proteins can provide information about their charge and size.

Quality Control (QC)

1. Consistency: Use pre-made gels or standardize gel preparation to ensure consistency between runs.

2. Standards and Controls:

   - Run molecular weight markers with each gel.

   - Include known protein samples as controls to verify the accuracy of the results.

3. Reagent Quality: Use high-purity reagents and fresh buffers to avoid contamination and degradation.

4. Equipment Maintenance: Regularly calibrate and maintain the electrophoresis apparatus and power supply.

5. Documentation: Record all experimental conditions, including buffer composition, gel percentage, voltage, and run time, to ensure reproducibility.

6. Replicates: Perform experiments in duplicates or triplicates to confirm the reliability of the results.

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