assay protocol

How many kinds of Native-PAGE are there by now?

1. Blue native PAGE ( BN-PAGE)

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein-protein interactions.Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection.

Refference: Wittig I, et al., Nat Protoc, 2006, 1(1):418-428.

2. Clear native PAGE(CN-PAGE)

Clear-native PAGE (CN-PAGE) separates acidic water-soluble and membrane proteins (pI < 7) in an acrylamide gradient gel, and usually has lower resolution than blue-native PAGE (BN-PAGE). The migration distance depends on the protein intrinsic charge, and on the pore size of the gradient gel. This complicates estimation of native masses and oligomerization states when compared to BN-PAGE, which uses negatively charged protein-bound Coomassie-dye to impose a charge shift on the proteins. Therefore, BN-PAGE rather than CN-PAGE is commonly used for standard analyses. However, CN-PAGE offers advantages whenever Coomassie-dye interferes with techniques required to further analyze the native complexes, e.g., determination of catalytic activities, as shown here for mitochondrial ATP synthase, or efficient microscale separation of membrane protein complexes for fluorescence resonance energy transfer (FRET) analyses. CN-PAGE is milder than BN-PAGE. Especially the combination of digitonin and CN-PAGE can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE. Enzymatically active oligomeric states of mitochondrial ATP synthase previously not detected using BN-PAGE were identified by CN-PAGE.

Refference: Wittig I and Schägger H, Proteomics, 2005, 5(17):4338-4346.

3. Quantitative preparative native continuous PAGE(QPNC-PAGE)

In QPNC-PAGE, the gel should be polymerized for about 69h. The pore size of the gel is large and therefore the molecular sieving action minimized in the electrophoretic separation. So the gel and the interaction between the active molecules can be ignored.

The metal protein to be isolated will not be decomposed into the apo protein and metal cofactor. The biological structure of the isolated protein (natural or 3D conformation) does not conformationally change after the QPNC-PAGE.

QPNC PAGE is applied to separate acidic, alkaline and neutral metal protein with molecular weight of 6-200kDa, and has important applications in the assays of relationship of structure and function of independent metal protein like blood or other clinical samples, because the incorrect metal may lead to protein misfolding.