Discovery and invention of Blue-Native Page (BN- PAGE)
BN- Page is the most efficient non-denaturing gel system which came up with the challenge of separating different kinds of membrane proteins in one step from both biological membranes and tissue or cell homogenates. This technique can be used in multiple areas like detection of protein masses and oligomeric states in their native form and identification of interaction between proteins in the physiological conditions (Wittig, Braun and Schägger, 2006). This technique was originally developed by Schägger and Jagow in 1991 to study the membrane proteins and multi-protein complexes (involved in oxidative phosphorylation system) within mitochondria in a single step (Schägger and von Jagow, 1991). They used mitochondria isolated from yeast or bovine heart and targeted the separation of protein complexes with molecular weights ranging between 10 kDa to 10 MDa (von Hagen, 2011).
The basic difference between a normal SDS-page and BN-Page is the addition of Coomassie dye G-250 to the cathode buffer of the system shortly before the run. It is an anionic dye which imparts negative charge to the proteins without denaturing them and retains protein-protein interactions. Membrane proteins are prevented from getting aggregated due to the nonspecific binding of dye to the hydrophobic areas of the proteins. The running pH of this technique is fixed at common pH, found within most of the intracellular compartments (7.5) under physiological conditions. This dye binds the membrane proteins uniformly (dye/protein ratio ~ 1g/g), due to which protein and protein complexes migrate towards the anode under the excessive force provided by the negative charge, hence getting separated within the gel based on their molecular mass (Krause, 2006).
Investigation of the bacterial protein complexes using BN-PAGE - Basic steps of the protocol
- Fractionation of the bacterial cell lysate into cytosol and membranes is done.
- Solubilization of the two compartments is done separately. Mild anionic detergents like Triton X-100, dodecylmaltosie or digitonin can be used for solubilization of membranes so that the detergent/protein ratio is 1g, 0.75g, 2 g for each detergent respectively (Hunte, 2003).
- Non denaturing conditions, which include, low salt concentration, neutral pH and absence of denaturing agent (SDS or urea) were maintained throughout during sample preparation and electrophoresis. Constant temperature was maintained (4 °C) and heating followed by freezing was avoided to prevent formation of artificial aggregates or disintegration of protein complexes (Dresler, Klimentova and Stulik, 2011).
- Coomassie Blue G-250 dye used during the process of electrophoresis was not directly added to the sample; rather it was allowed to be released from the cathode buffer. Due to this proteins having pI below 7 migrated towards anode and the bulk detergent remained in the sample well.
- A non-colored sample is loaded into the sample well. The maximum permissible limit for sample loading is 200μg of protein into a 1x 0.16 cm sample well.
- The electrophoresis is stopped as soon as the desired protein band is detected. The blue bands of the desired protein are extracted into a cathodic arm of an electroelutor through a syringe (Hunte, 2003).
- 2 dimensional BN/SDS-PAGE is used latter to separate proteins belonging to the same native protein complex (Dresler, Klimentova and Stulik, 2011).
Applications of BN - PAGE
BN - PAGE is used in a large number of experiments like:
1. Analysis of protein complexes present within organelles and prokaryotes- This technique has emerged as a primary tool to analyze the protein complexes within mitochondria, ER or those involved in the protein import within yeast etc. Even the transient super complexes like TOM-TIM-preprotein and protein complexes from prokaryotes (photosynthetically active bacteria and Agrobacterium strains) can be detected using this technique (Krause, 2006).
2. Analysis and detection of protein-protein interactions - Most of the detergents used in BN-page are gentle and maintain the integrity of the biological samples hence retaining the protein-protein interactions. The size of the protein is determined by its mobility; hence this technique can be used to determine the oligomeric state of the proteins (ex - glycoprotein), stoichiometry of respiratory supercomplexes and determination of monomer, dimer subunits of a homo-supercomplex (ex: mitochondrial ATP synthases). This is the best method to isolate and characterize the proteins in their native state which can be proved by the retention of the enzymatic activity of the protein complexes obtained after electroelution. Identification of catalytic or structural defects of OXPHOS complexes through BN-page helped in the clinical diagnosis of OXPHOS diseases. Low abundance protein complexes, which can usually be detected through radiolabelling can be identified with the help of Antibody-shift BN-PAGE. A combination of BN-PAGE and radiolabelling of proteins helps to examine various assembly steps involved in the formation of protein complexes in a time dependent manner (Krause, 2006).