Development of Blue Native Gel Electrophoresis Protocol for the Analysis of Properdin Oligomers in Lupus Serum

Abstract

SLE is an autoimmune illness exhibiting great heterogeneity possessing great challenges upon clinical investigators and healthcare professionals. The pathogenesis exhibits a hinge between an excessive sustained autoantibody generation and loss of immune tolerance. Properdin is recognized as the only known positive regulator of alternative complement activation pathway that is hyperactivated throughout SLE. Properdin is suggested to be linked to lupus severity since reduced symptoms were seen in properdin-deficient MRL/Ipr mice. Circulating protein exists as oligomeric mixture of dispersed polymers whose functional activity increases with polymers respective sizes. Here, the adopted two-dimension separation protocol (1D BN-PAGE / 2D SDS-PAGE) and Western blotting immunodetection techniques have been successful for identification of properdin oligomers in mice sera. It was clear that conducting the gradient SDS-PAGE technique under non-reducing conditions as well as treating the 1D BN-PAGE slabs with dissociating solution was beneficial for properdin oligomer resolution. Therefore, the adopted protocol can now be applied to study lupus-prone and wildtype sera in parallel, while as adding to the knowledge of understanding the SLE pathogenesis addressing severity/risk. Although detection of properdin monomer bands could question the technique performance for investigating native serum properdin, the small sample size and detection of two properdin oligomers could ensure the technique beneficial over other ones including EM, NMR, immunodiffusion, and immunoprecipitation. Future work for fine tuning the proposed technique performance as well as quantifying and identifying the properdin oligomers within human and properdin-deficient MRL/Ipr mice samples would be worthy of investigation