RESUMO
We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.
Assuntos
Western Blotting/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida , Colódio/isolamento & purificação , Glicoproteínas de Membrana/química , Proteínas de Membrana/química , Membranas Artificiais , Dados de Sequência Molecular , Peptídeos/química , Proteínas/química , Soroalbumina Bovina , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Uroplaquina II , Uroplaquina IIIRESUMO
Based on successful preliminary bench scale experimental studies for treatment of industrial wastewater contaminated by nitrocellulose, a pilot plant is constructed for results assessment. Bench scale experimental work proved 55%, 73% recovery of nitrocellulose without and with chemical addition respectively within 10 min flotation compared to 35%, 69% recovery within 2-0.5h settling respectively. The treatment process aims the recovery of nitrocellulose through an efficient dissolved air flotation (DAF) unit. Different operating conditions have been studied for different effluent characteristics with and without flocculating agent. Nitrocellulose recovery reached 80% by flotation without chemical, which is increased to 87% using cationic polymer, but both exploring suitable pathways to solve the recovery problem.The experimental results are considered suitable basis for full scale design of the industrial treatment unit.