Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies

Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies
March 2004
Received 1 March 2004; accepted 8 March 2004. Available online 2 April 2004
Carmen Sara Hern?ndez, Ana Rodrigo and Juan Ferr?
Journal of Invertebrate Pathology
Volume 85, Issue 3 , March 2004, Pages 182-187
ScienceDirect
Departament de Gen?tica, Universitat de Val?ncia, 46100, Burjassot (Valencia), Spain
Abstract
Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors. Collaboration between laboratories often require that frozen insect samples are sent in dry ice. Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping. We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B. thuringiensis Cry toxins. For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua, Manduca sexta, and Helicoverpa armigera. Higher membrane protein recovery was always obtained from lyophilized midguts compared to frozen midguts, and similar membrane marker enzyme activities were found in BBMV from either treatment. Comparable equilibrium dissociation constants and binding site concentrations, calculated from binding experiments with labeled 125I-Cry1Ab toxin, were found using BBMV from either method. In the light of these results, lyophilization is a good preserving method of lepidopteran midguts to study binding of B. thuringiensis Cry toxins.
Author Keywords: Tissue preservation; Cry toxins; BBMV; Membrane receptors