De 52

The mung bean seeds were obtained from Sri Venkateswara Agricultural University, Tirupati. Soybean LOX, ETYA, Linoleic acid, Linolenic acid, Arachidonic acid, Sephadex G-100, DE-52, Poly Buffer Exchangers 94 (PBE-94), Phenyl methyl sulfonyl fluoride (PMSF), EDTA, Acrylamide, Bis-Acrylamide, Coomassie brilliant blue, Lauryl sulphate (SDS) and protein size markers were procured from Sigma Chemicals Co (St. Louis, MO, USA). NDGA was a generous gift from department of chemistry, S.V. University. All other chemicals were reagent grade procured from Merck, Mumbai, India.

Dissected tick salivary glands were collected in a tube with 2 ml of cold PBS, transferred to a chilled glass hand homogenizer (‘UNI-FORM’ (England), 4–5 ml of homogenizer) and disrupted by gentle up, down and circular motion of the pestle until a uniform cloudy suspension was formed. The suspension was then centrifuged at 1000g at 4 °C for 5 min. The resulting supernatant containing sporozoites was applied to an 8 ml diethylaminoethyl cellulose (DE-52; Sigma–Aldrich (USA) Cat. No. D3764) column (Musoke et al., 1992 (link)). Flow-through fractions of 3.5 ml were collected in siliconised 10 ml conical bottomed glass tubes on ice. The sporozoite-rich cloudy fractions (numbers 2–5) were pooled and centrifuged at 12,000g at 4 °C for 5 min. The resulting sporozoite-rich small yellowish pellet was resuspended in SDS–PAGE sample buffer and heated at 100 °C for 5 min prior to fractionation on 10% one-dimensional SDS–PAGE and staining with Coomassie Blue.

STZ, DE-52, insulin ELISA kit and p-NCS salt were purchased from Sigma-Aldrich (Beirut office Lebanon). p-NCS was prepared as a substrate reagent for ASA ASB as previously explained [28 (link)]. CAT assay kit was purchased from Biodiagnostic, Diagnostic and Research Reagents, Giza, Egypt.