For this week's "Antibody Applications" series, we're delighted to have our first guest writer, Eric Torres, tell us about Blue Native-PAGE.
Eric is a PhD Candidate in Biochemistry and Molecular Biology at the University of California, Los Angeles, working in the lab of Dr. Carla Koehler. Take it away Eric!
BN-PAGE or Blue Native Polyacrylamide Gel Electrophoresis is a common and inexpensive technique to resolve protein complexes by molecular weight while retaining their native structure through gel electrophoresis.
BN-PAGE acts by using Coomassie Blue-G250 dye to coat proteins with the necessary negative charge for migration to the anode. This contrasts with the more conventional SDS-PAGE which uses the strong ionic detergent SDS to sort individual proteins based on their charge/mass ratio.
Instead, protein complexes migrate across the gel according to their specific pore size in acrylamide gradient gels until they have reached their specific pore size limit. This is because the anionic character, solubility in water and ability to bind to membrane proteins of the Coomassie Blue-G250 allows it to be sufficient substitute for SDS.
BN-PAGE can separate protein complexes ranging between 100 kDa to 10 MDa and can be adjusted depending on the concentration range of acrylamide in the gradient gel. One of the most important things to consider is the type of lysate and detergent to maintain the specific complex or protein-protein interaction being studied. Typically, a mild detergent such as digitonin or dodecylmaltoside are used for BN-PAGE, though low concentrations of Triton X-100 have also been used.
BN-PAGE was popularized in the early 90s to characterize respiratory supercomplexes of the mitochondrion. Supercomplexes of the electron transport chain can be can be easily detected by a simple Coomassie stain from a BN-PAGE.
Since then, BN-PAGE has been used for diverse applications including clinical diagnosis, assessing stochiometric amounts of native complexes and identifying protein-protein interactions. BN-PAGE can also be coupled with a wide range of downstream protein detection methods including silver stain, immunoblot, mass spectrometry and radiolabeled protein labeling.
A limitation for BN-PAGE is that it requires clean and robust antibodies that can detect the protein in its native form. Antibodies based on denatured antigens of the protein may have trouble detecting proteins on a BN-PAGE. If a clean and robust antibody isn’t available, a second dimension can be added known as a 2D-SDS PAGE (more on 2D-SDS PAGE in the next post).
Lack of resolution between protein complexes may also be an issue and would require troubleshooting the gradient gel parameters. Furthermore, the Coomassie dye isn’t completely inert to protein-protein interactions, causing some disruption of native complexes and the presence of salts or other solutes can cause protein smearing. In these cases, a CN-PAGE or Colorless Native gel that lacks the anionic G-250 dye may be preferred.
Despite certain limitations, BN-PAGE remains a staple technique for molecular biology labs because it is inexpensive, requires no specialized equipment and compatibility with most protein detection methods.
In the next article, I will address the technical details and nuances of running a BN-PAGE.
For more in-depth discussion on BN-PAGE, please refer to Wittig et. al 2006 in Nature Protocol, and this illustration (kindly provided by Reddit user Natolx).