Polyacrylamide Gel Electrophoresis (PAGE) – Principle, Procedure, Applications, Advantages & Limitations

Polyacrylamide Gel Electrophoresis (PAGE) – Principle, Procedure, Applications, Advantages & Limitations

by

Introduction to Polyacrylamide Gel Electrophoresis

  • Electrophoresis is a standard method used in molecular biology and biochemistry to separate, identify, and analyze biomolecules like proteins and nucleic acids.
  • The separation is achieved by forcing molecules to migrate through a porous gel matrix under an electric field.
  • Two main gel types are used: agarose gel and polyacrylamide gel.
  • Polyacrylamide Gel Electrophoresis (PAGE) provides greater resolution than agarose gels, especially for proteins and small DNA fragments.
  • PAGE is widely used in molecular biology, genetics, forensic science, biochemistry, and biotechnology.

In simple terms: PAGE is a laboratory technique used to separate proteins and nucleic acids based on their size and charge by making them move through a polyacrylamide gel under electricity.

What is Polyacrylamide Gel?

  • Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent such as N,N’-methylenebisacrylamide.
  • The polymerization is initiated by ammonium persulfate (APS) and catalyzed by TEMED (N,N,N’,N’-tetramethylethylenediamine).
  • The pore size of the gel depends on the ratio of acrylamide to bisacrylamide, making it highly adjustable for separating molecules of different sizes.
  • This makes polyacrylamide gels superior to agarose gels when working with small proteins or DNA fragments.

Principle of PAGE

The principle of PAGE is based on two main concepts:

  1. Electrophoretic Mobility
    • Charged molecules migrate in an electric field towards the electrode with the opposite charge.
    • The rate of migration depends on charge, size, and shape of the molecule.
  2. Denaturation for Size-Based Separation
    • Different proteins naturally have different shapes and charges, which makes comparison difficult.
    • To overcome this, proteins are treated with SDS (Sodium Dodecyl Sulfate), which:
      • Denatures proteins (removes secondary, tertiary, quaternary structures).
      • Covers them with a uniform negative charge.
    • Thus, the separation depends only on size (molecular weight).

In summary: In SDS-PAGE, proteins move through the gel at different speeds based on their molecular size, with smaller proteins migrating faster.

Requirements for PAGE

To perform PAGE, the following materials are required:

  • Acrylamide and Bisacrylamide solution (for resolving and stacking gels).
  • Gel loading buffer (contains tracking dye and denaturants).
  • Running buffer (commonly Tris-Glycine buffer).
  • Staining and destaining solutions (Coomassie Brilliant Blue, silver stain, or ethidium bromide for nucleic acids).
  • Protein or nucleic acid samples.
  • Molecular weight markers (protein ladders) for comparison.
  • Electrophoresis equipment:
    • Gel casting stand and glass plates
    • Combs for sample wells
    • Electrophoresis chamber and power supply

Steps in Polyacrylamide Gel Electrophoresis (PAGE)

1. Sample Preparation

  • Protein samples are mixed with SDS to denature and impart uniform negative charge.
  • A reducing agent (like β-mercaptoethanol or DTT) may be added to break disulfide bonds.
  • Samples are heated (~60–100°C) to ensure full denaturation.
  • Tracking dye is added to monitor electrophoresis progress.
Steps in Polyacrylamide Gel Electrophoresis; Sample Preparation
Figure: Steps in Polyacrylamide Gel Electrophoresis; Sample Preparation

2. Preparation of Polyacrylamide Gel

  • Gel consists of acrylamide + bisacrylamide, APS, TEMED, and buffer.
  • Two layers are prepared:
    • Resolving gel: Higher % acrylamide (5–25%) for separating proteins.
    • Stacking gel: Lower % acrylamide to concentrate samples before separation.
  • Gel is cast between two glass plates and wells are created with combs.
Polyacrylamide Gel Electrophoresis; Preparation of Polyacrylamide gel
Figure: Polyacrylamide Gel Electrophoresis; Preparation of Polyacrylamide gel

3. Loading Samples and Running the Gel

  • After polymerization, wells are filled with samples and a molecular weight marker.
  • The gel is placed in the electrophoresis chamber filled with buffer.
  • Electric current is applied: negatively charged molecules migrate towards the anode.
  • Smaller proteins migrate faster through the pores, larger ones slower.
Polyacrylamide Gel Electrophoresis; Loading Samples and Running the Gel
Figure: Polyacrylamide Gel Electrophoresis; Loading Samples and Running the Gel

4. Detection/Visualization

  • After electrophoresis, gels are stained to visualize separated molecules.
  • Common stains:
    • Coomassie Brilliant Blue (routine protein staining).
    • Silver stain (high sensitivity).
    • Ethidium bromide or SYBR Green (for nucleic acids).
  • After staining, bands appear at different positions corresponding to proteins/DNA of different sizes.

Applications of PAGE

  1. Protein Analysis
    • Estimation of molecular weight.
    • Determination of protein purity.
    • Study of protein subunits and quaternary structures.
    • Peptide mapping.
  2. Nucleic Acid Studies
    • Analysis of small DNA or RNA fragments.
    • Detection of mutations or polymorphisms.
  3. Medical and Clinical Uses
    • Diagnosing protein abnormalities.
    • Analyzing hemoglobin variants.
    • Studying enzyme deficiencies.
  4. Research and Biotechnology
    • Used before Western blotting.
    • Quality control in recombinant protein production.
    • Comparing protein composition between samples.
  5. Forensic Applications
    • Identification of proteins/DNA in criminal investigations.

Advantages of PAGE

  • High resolving power – sharp, distinct bands.
  • Adjustable pore size by changing acrylamide concentration.
  • Can separate proteins of similar size effectively.
  • Produces highly pure DNA or protein fragments.
  • Works well for low molecular weight proteins.

Disadvantages of PAGE

  • Preparation is more complex than agarose gels.
  • Acrylamide is toxic, requiring careful handling.
  • Gels are less reusable – a new gel must be prepared for each run.
  • More time-consuming and prone to technical errors.

Types of PAGE

  1. SDS-PAGE (Denaturing PAGE)
    • Most common type.
    • Proteins denatured with SDS → separation based only on size.
  2. Native PAGE (Non-denaturing PAGE)
    • Proteins retain native structure and activity.
    • Separation depends on charge, shape, and size.
  3. 2D-PAGE (Two-dimensional PAGE)
    • Combines isoelectric focusing (IEF) and SDS-PAGE.
    • Used for complex proteomic studies.

Conclusion

  • Polyacrylamide Gel Electrophoresis (PAGE) is one of the most important molecular biology techniques for analyzing proteins and nucleic acids.
  • With variations like SDS-PAGE, Native PAGE, and 2D-PAGE, it plays a crucial role in diagnostics, proteomics, clinical studies, and biotechnology research.
  • Despite being labor-intensive and involving toxic chemicals, its accuracy and resolution make PAGE a gold standard in protein analysis.

Frequently Asked Questions (FAQs) on Polyacrylamide Gel Electrophoresis (PAGE)

Q1. What is Polyacrylamide Gel Electrophoresis (PAGE)?
Ans: PAGE is a laboratory technique used to separate proteins and nucleic acids by moving them through a polyacrylamide gel under an electric field.

Q2. Why is polyacrylamide used instead of agarose?
Ans: Polyacrylamide has smaller, adjustable pores, giving higher resolution for separating proteins and small DNA fragments compared to agarose.

Q3. Who introduced PAGE?
Ans: The technique was developed in the 1950s and 1960s by scientists studying protein separation, later refined into SDS-PAGE by Ulrich K. Laemmli in 1970.

Q4. What is the principle of PAGE?
Ans: Molecules migrate in an electric field at different speeds depending on charge, size, and shape. In SDS-PAGE, separation is based mainly on molecular weight.

Q5. What are the types of PAGE?
Ans:

  • SDS-PAGE (denaturing) → size-based separation.
  • Native PAGE → separation based on charge, size, and shape (proteins remain active).
  • 2D-PAGE → combines isoelectric focusing (pH separation) with SDS-PAGE.

Q6. What is SDS-PAGE?
Ans: SDS-PAGE uses sodium dodecyl sulfate (SDS) to denature proteins and give them a uniform negative charge, so migration depends only on size.

Q7. What is Native PAGE?
Ans: In Native PAGE, proteins are not denatured. They retain their structure and activity, and separation depends on charge + size + shape.

Q8. What is the role of the stacking gel in PAGE?
Ans: The stacking gel has low acrylamide concentration and helps concentrate all proteins into sharp bands before entering the resolving gel.

Q9. What is the function of TEMED in PAGE?
Ans: TEMED (Tetramethylethylenediamine) catalyzes the polymerization of acrylamide into polyacrylamide gel.

Q10. Why is acrylamide considered hazardous?
Ans: Acrylamide monomers are toxic and neurotoxic; they must be handled with gloves and safety precautions.

Q11. What stains are used to visualize proteins in PAGE?
Ans: Common stains:

  • Coomassie Brilliant Blue (routine staining)
  • Silver stain (high sensitivity)
  • SYBR Green / Ethidium Bromide (for nucleic acids)

Q12. Can PAGE be used for DNA and RNA?
Ans: Yes. PAGE can separate small DNA and RNA fragments, but agarose gels are preferred for large DNA fragments.

Q13. What is the difference between PAGE and agarose gel electrophoresis?
Ans: PAGE → high resolution, best for proteins & small DNA.
Agarose gel → best for large DNA fragments (hundreds of bp to kb).

Q14. What is the role of SDS in PAGE?
Ans: SDS denatures proteins and binds uniformly, giving all proteins a negative charge proportional to their length, making separation purely size-based.

Q15. What is 2D-PAGE?
Ans: Two-dimensional PAGE first separates proteins by isoelectric focusing (based on charge) and then by SDS-PAGE (based on size). Used in proteomics.

Q16. How is PAGE used in medicine?
Ans: PAGE is used to detect abnormal blood proteins, hemoglobin variants, and enzyme deficiencies, aiding in disease diagnosis.

Q17. How is PAGE used in biotechnology?
Ans: PAGE helps in protein purification, Western blotting, recombinant protein analysis, and quality control.

Q18. What are the advantages of PAGE?
Ans:

  • High resolution.
  • Adjustable pore size.
  • Sharp bands for accurate analysis.
  • Useful for low molecular weight proteins.

Q19. What are the disadvantages of PAGE?
Ans:

  • Complex preparation compared to agarose.
  • Acrylamide is toxic.
  • Gel is single-use.
  • Time-consuming.

Q20. Why is PAGE important in research?
Ans: PAGE is a fundamental technique in molecular biology for analyzing proteins, nucleic acids, and is essential for proteomics, clinical studies, and biotechnology.

Q21. What is a protein ladder in PAGE?
Ans: A protein ladder (molecular weight marker) is a mixture of proteins of known sizes used to estimate the size of unknown proteins in a sample.

Q22. Can PAGE be quantitative?
Ans: Mostly qualitative/semi-quantitative. However, with densitometry analysis, PAGE results can provide quantitative information.

Q23. What is the difference between SDS-PAGE and Western blotting?
Ans: SDS-PAGE separates proteins by size.
Western blotting transfers separated proteins to a membrane and uses antibodies for specific protein detection.

References

  1. http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch07s03.html
  2. https://www.wou.edu/las/physci/ch462/Gel%20Electrophoresis.pdf
  3. https://microbenotes.com/polyacrylamide-gel-electrophoresis-page/
  4. https://www.slideshare.net/mbn1994/introduction-principle-instrumentation-and-applications-of-sdspage-55728195
  5. https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis
  6. https://msu.edu/course/css/451/Lecture/PT-electrophoresis%20(2009).pdf
  7. http://library.umac.mo/ebooks/b28050459.pdf

Leave a Comment