Native PAGE

Unlike SDS-PAGE, native (non-denaturing) PAGE separates proteins in their native conformation without SDS or reducing agents. The migration rate depends on both the protein’s size and its intrinsic charge, making molecular weight estimation less straightforward than in SDS-PAGE — but preserving enzymatic activity and protein–protein interactions.

Buffers: The most common system is the Laemmli buffer system without SDS. The resolving gel is at pH 8.8 and the stacking gel at pH 6.8, both with Tris-glycine running buffer. Proteins carry a net negative charge at the running pH and migrate toward the anode.

Blue Native PAGE (BN-PAGE): Coomassie Blue G-250 is added to the cathode buffer and the protein sample. The dye binds to hydrophobic surfaces, imparting a uniform negative charge without denaturing the proteins. This allows separation of membrane protein complexes (e.g., respiratory chain supercomplexes) that are insoluble under standard native conditions.

Applications:

• Determining the oligomeric state of proteins (monomer, dimer, tetramer).
• Analyzing multi-protein complexes by Western blot or mass spectrometry after in-gel digestion (native-PAGE followed by 2D SDS-PAGE).
• Detecting enzyme isoforms with different electrophoretic mobilities.
• Quality control for protein purification (aggregation assessment).

Limitations: resolution is generally lower than SDS-PAGE. Acidic and basic proteins co-migrate poorly in the same gel system. Calibration with native molecular weight markers gives only approximate sizes.

Zymography

Zymography detects enzyme activity directly in a polyacrylamide gel by co-polymerizing a substrate into the resolving gel. After electrophoresis, the gel is incubated in a renaturation buffer to restore enzyme activity, then stained. Enzyme activity appears as a clear band against a dark background where the substrate has been degraded.

Types of zymography:

• Gelatin zymography: gelatin (denatured collagen) is incorporated at 0.1%. Used for matrix metalloproteinases (MMPs). After incubation, the gel is stained with Coomassie Blue — MMP activity appears as white/transparent bands on a blue background.
• Casein zymography: casein is the substrate. Used for serine proteases and plasminogen activators.
• Reverse zymography: the gel contains both the substrate and an inhibitor. Inhibitor activity appears as dark bands where the substrate is preserved against degradation.
• In-gel kinase assays: the gel is polymerized with a protein kinase substrate, and after electrophoresis, renaturation, and incubation with [γ-³²P]ATP, phosphorylated bands are detected by autoradiography.

Protocol summary for gelatin zymography:

1. Prepare the resolving gel with 0.1% gelatin (and SDS).
2. Load samples under non-reducing conditions (no boiling, no reducing agents — these denature MMPs).
3. Run at 4 °C to prevent premature proteolysis.
4. Remove SDS by washing the gel in 2.5% Triton X-100 for 30 minutes (2 times).
5. Incubate in developing buffer (50 mM Tris, 10 mM CaCl₂, 0.15 M NaCl, pH 7.5) at 37 °C for 16–48 hours.
6. Stain with Coomassie Blue and destain.

Zymography is highly sensitive (detecting picogram amounts of active enzyme) and can distinguish between pro-enzyme (latent) and active forms based on molecular weight.