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Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases.
The gut extract was incubated at room temperature on a rotator shaker with 0.5 mg/ml Micrococcus lysodeikticus cells prepared in same buffer for 24 h.
Coverslips were dipped into liquid nitrogen and removed as quickly as possible (less than 1 second) and abalone gut extract was omitted from the enzyme mixture.
∼60 μg protein for the adult gut extract, or ∼250 μg protein for the whole larvae extract was added to 700-μl reaction buffer (50 mM K2HPO4, pH 7.4, containing 30 µM citric acid).
The fly gut extract (0.75 µg−1) was pre-incubated with increasing concentrations of rISP1 (0.7 µM–7 µM) in 100 mM Tris-HCL pH 8.0, 100 mM NaCl, 2.5% (v/v) DMSO, for 5 min at 4°C.
For ISP2, the fly gut extract (0.5 µg−1) was pre-incubated with increasing concentrations of rISP2 (2 nM–2 µM) in 100 mM Tris-HCl pH 8, 100 mM NaCl, for 5 min at 4°C.
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Enzymatic activities against carboxymethylcellulose (CMC), xylan and pectin have already been described for whole gut extracts from P. cochleariae larvae [ 21].
Interestingly these unknown microorganisms were found most often in guts extracted from larvae that fed on ir defensin (76 and 96) plants.
Oeggl, K. et al. The reconstruction of the last itinerary of "Ötzi", the Neolithic Iceman, by pollen analyses from sequentially sampled gut extracts.
Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities.
Crude gut extracts were also prepared from larvae exposed to ButaIT/GNA diets to verify binding of the fusion protein to the gut epithelium.
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