Proteomics & 2D Gels

Explain the principle of isoelectric focusing (IEF) in 2D gel electrophoresis and how it separates proteins.

how does it separate proteins

based on their isoelectric point (pI),

isoelectric point (pI),

the pH at which a protein has no net charge.

principles of Isoelectric Focusing (IEF) in 2D Gel Electrophoresis:

1. pH Gradient:

2. Protein Migration:

   1. Focusing:

1. pH Gradient:

A stable pH gradient is established using immobilized pH gradient (IPG) strips,

contain covalently bound buffering groups (e.g., Immobiline™).

1. Protein Migration:

electric field is applied,

proteins migrate through the gradient.

1. Protein Migration:

In a pH < pI,

proteins are positively charged

move toward the cathode

1. Protein Migration:

in a pH > pI,

proteins are negatively charged

move toward the anode.

1. Focusing:

Proteins stop migrating at their pI,

their net charge is zero,

forming sharp bands.

Separation Outcome:

Proteins are resolved along the strip according to their pI values.

Combined with the second dimension (SDS-PAGE,

2D gels provide high-resolution separation of complex protein mixtures.

SDS-PAGE, which separates by

molecular weight),

Limitations of 2D Gel Electrophoresis for Proteomic Analysis:

1. Poor Resolution of Hydrophobic and Low-Abundance Proteins:

   1. Reproducibility and Technical Challenges:

1. Poor Resolution of Hydrophobic and Low-Abundance Proteins:

   • Membrane proteins (hydrophobic) often

• aggregate during IEF, leading to their underrepresentation.

1. Hydrophobic and Low-Abundance Proteins:

   • Low-abundance proteins may be

• obscured by high-abundance proteins (e.g., albumin in serum), limiting detection sensitivity.

1. Reproducibility and Technical Challenges:

   • Variability in ?can make gel comparisons difficult.

• pH gradients, sample loading, and staining

1. Reproducibility and Technical Challenges:

   • ? compared to liquid chromatography-mass spectrometry (LC-MS).

• Labor-intensive and time-consuming, with limited scalability for high-throughput studies

Impact: These limitations drove

the adoption of complementary techniques like MudPIT (multidimensional protein identification technology) for comprehensive proteome coverage.

How does the TAP (Tandem Affinity Purification) tag method improve protein complex isolation compared to traditional affinity chromatography?

two-Step Purification for Higher Specificity:

Mild Elution Conditions Preserve Native Complexes:

Reduced Background Contamination:

Two-Step Purification for Higher Specificity:

• The TAP tag combines

• Protein A (binds IgG) and Calmodulin-Binding Peptide (CBP) in tandem,

  • separated by a TEV protease cleavage site.

Two-Step Purification for Higher Specificity:

• First step:

• IgG beads capture the tagged protein and its interactors.

Two-Step Purification for Higher Specificity:

• TEV protease elution

• removes nonspecifically bound proteins while preserving the complex.

Two-Step Purification for Higher Specificity:

• Second step:

The eluate is applied to calmodulin beads (Ca²⁺-dependent), and gentle EGTA elution minimizes disruption of weak interactions.

Two-Step Purification for Higher Specificity: steps

IgG beads capture the tagged protein and its interactors.

• TEV protease elution removes nonspecifically bound proteins while preserving the complex.

The eluate is applied to calmodulin beads (Ca²⁺-dependent), and gentle EGTA elution minimizes disruption of weak interactions.

Mild Elution Conditions Preserve Native Complexes:

• Traditional methods often use

• harsh elution (e.g., low pH or high salt), which can dissociate fragile complexes.

Mild Elution Conditions Preserve Native Complexes:tAP employs

protease cleavage (TEV) and Ca²⁺ chelation (EGTA), maintaining near-physiological conditions.

1. Reduced Background Contamination:

   • The sequential purification steps

• drastically lower nonspecific binding, yielding cleaner samples for MS analysis.