A simple method for immunodetection of membrane‐associated proteins

Abstract

The main objective of the present study was to identify membrane antigenic proteins using immunodiffusion and Western blotting techniques. This multidisciplinary experiment integrates biochemical principles with immunological and microbiological concepts. Pasteurella multocida, the causative agent of fowl cholera and other diseases like haemorrhagic septicaemia in many wild and domestic animals, was chosen as a biological model in order to obtain membrane antigenic proteins. We designed some simple laboratory experiments: partial purification of IgG, study of the effect of detergent on immunodiffusion, electrophoresis and Western blotting of membrane protein fraction of Pasteurella multocida. The results obtained with these techniques revealed that the membrane antigenic proteins Pasteurella multocida presented had Mr's of around 110, 104, 86, 71, 65 and 40kDa. © 2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

Digital Image Analysis to Standardize a Photometric Method in Colorimetric Quantification

“…[5] Bovine serum albumin (BSA) was used in known concentrations to obtain standard curves for determination of unknown concentration of the membrane protein of Pasteurella mutocida bacteria [6] and the Na,K-ATPase, from rabbit kidney. [7] These substances served as unknown concentration protein samples.…”

Sistemas carreadores de proteínas antigênicas da membrana de Pasteurella multocida para a prevenção da pasteurelose

“…Foi realizada uma ultracentrifugação a 140.000 x g por 1 hora, a temperatura ambiente, para o isolamento das frações de membrana. Finalmente, o pellet foi ressuspenso em 25 mL do tampão Tris-HCl e armazenado em alíquotas de 1 mL, em freezer a -20 o C (Daghastanli et al, 2001;Daghastanli et al, 2004).…”

“…O anticorpo contra a P. multocida foi obtido pela inoculação intramuscular de 1 mL da bactéria inativada, preparada conforme descrito no item 3.2, contendo aproximadamente 10 9 ufc, em coelhos saudáveis sem distinção de sexo e com reforço semanal durante um 1 mês. O soro total dos animais foi coletado 75 dias após a primeira imunização (Daghastanli et al, 2001).…”