Two-Dimensional Blue Native/SDS Polyacrylamide Gel Electrophoresis for Analysis of Brazilian Bothrops Snake Venoms

Abstract

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

Crosslinked modified decellularized rabbit conjunctival stroma for reconstruction of tissue-engineered conjunctiva in vitro

“…After trying a series of decellularization protocols, we eventually developed a PLA2 decellularized method for conjunctival tissue regeneration. PLA2 is the low molecular mass enzymes (13-16 kDa) that specially hydrolyze phospholipid [16], and exhibit no enzymatic reaction site on the collagen that ECM were well preserved (figure 4). There was a significant decrease in the DNA of renal cells treated with decellularization, and the CCK-8 results indicated that the decellularized tissues did not reduce chondrocyte proliferation compared to the control group (figures 6(A) and (B)).…”