Methods of cell fractionation, electrophoresis of nucleic acids and proteins.

Understanding the structure and function of cells and biomolecules often requires isolating specific cellular components or analyzing nucleic acids and proteins. Two essential laboratory techniques used for these purposes are **cell fractionation** and **electrophoresis**.

Cell fractionation

Cell fractionation is the process of breaking open cells and separating their components based on size, density, and other physical properties. It allows researchers to study organelles and macromolecules in isolation.

General steps

1. **Cell disruption (homogenization)**

• Cells are lysed using mechanical (blender, homogenizer), chemical (detergents), or enzymatic methods.
• The goal is to break membranes while preserving organelles.

1. **Differential centrifugation**

• The homogenate is subjected to increasing centrifugal forces.
• Components are separated based on size and sedimentation rate.

Centrifugal force	Pellet contains
600 × g	Nuclei, unbroken cells
10,000 × g	Mitochondria, lysosomes, peroxisomes
100,000 × g	Microsomes, plasma membrane fragments
>100,000 × g	Ribosomes, soluble proteins

1. **Density gradient centrifugation**

• Organelles are further purified using a sucrose or Percoll density gradient.
• Each component migrates to its buoyant density.

Applications

• Study of mitochondrial function
• Lysosomal enzyme assays
• Isolation of ribosomes or nuclei for genetic analysis

Electrophoresis

Electrophoresis is a method used to separate macromolecules such as DNA, RNA, or proteins based on size and/or charge by applying an electric field.

Electrophoresis of nucleic acids

1. Agarose gel electrophoresis

• Used for separating DNA and RNA fragments (typically >100 bp).
• DNA is negatively charged and migrates toward the positive electrode.
• Separation is based on **fragment size**—smaller fragments migrate faster.

• **Steps:**

1. DNA is loaded into wells of an agarose gel.
2. An electric current is applied.
3. DNA bands are visualized using ethidium bromide or SYBR Green under UV light.

• **Applications:**
  • PCR product analysis
  • Restriction fragment analysis
  • DNA integrity testing

2. Polyacrylamide gel electrophoresis (PAGE) for nucleic acids

• Higher resolution than agarose.
• Used for small RNA fragments or sequencing gels.

Electrophoresis of proteins

1. SDS-PAGE (Sodium dodecyl sulfate PAGE)

• Denatures proteins and gives them a uniform negative charge.
• Separation is based on **molecular weight**.

• **Steps:**

1. Proteins are denatured with SDS and a reducing agent (e.g., β-mercaptoethanol).
2. Samples are loaded into a polyacrylamide gel.
3. An electric current separates proteins by size.
4. Proteins are stained (e.g., Coomassie Brilliant Blue, silver stain).

• **Applications:**
  • Protein purity and size determination
  • Expression analysis
  • Western blotting

2. Native PAGE

• Proteins are not denatured—preserves their native structure and charge.
• Separation depends on size, shape, and charge.

3. Isoelectric focusing (IEF)

• Proteins are separated based on **isoelectric point (pI)**.
• Proteins migrate in a pH gradient until they reach a pH where their net charge is zero.

4. 2D electrophoresis

• Combines IEF (first dimension) with SDS-PAGE (second dimension).
• Separates complex protein mixtures by pI and molecular weight.

Summary table

Method	Target	Principle
Differential centrifugation	Organelles	Sedimentation rate (size/density)
Agarose gel electrophoresis	DNA/RNA	Size-based separation
SDS-PAGE	Denatured proteins	Size (molecular weight)
Native PAGE	Native proteins	Size + charge + shape
Isoelectric focusing	Proteins	Isoelectric point
2D electrophoresis	Proteins	pI + size

References

Related articles

• Centrifugation
• DNA electrophoresis
• Protein electrophoresis
• SDS-PAGE
• DNA
• RNA
• Protein structure

Literature

• ALBERTS, Bruce. Molecular Biology of the Cell. 6. edition. New York : Garland Science, 2015. 483–499 pp. ISBN 978-0-8153-3218-3.

• BERG, Jeremy M. – TYMOCZKO, John L.. Biochemistry. 8. edition. New York : W.H. Freeman, 2015. 88–99 pp. ISBN 978-1-4641-2610-7.