Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies.
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 (125)I-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.
Hernández
Carmen Sara
CS
Departament de Genètica, Universitat de València, 46100 Burjassot (Valencia), Spain.
Rodrigo
Ana
A
Ferré
Juan
J
D000818:Animals; D001413:Bacillus thuringiensis / Q000502:physiology*; D001426:Bacterial Proteins; D015925:Cryopreservation; D004064:Digestive System / Q000469:parasitology*; D005612:Freeze Drying; D006790:Host-Parasite Interactions / Q000502:physiology; D019476:Insect Proteins / Q000378:metabolism*; D007915:Lepidoptera / Q000469:parasitology* / Q000502:physiology; D008871:Microvilli / Q000378:metabolism; D010572:Pest Control, Biological; D011956:Receptors, Cell Surface / Q000378:metabolism*
D001426:Bacterial Proteins; C095416:Cry toxin receptors; D019476:Insect Proteins; D011956:Receptors, Cell Surface