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  • Enzymatic Assays and Enzyme Histochemistry ofTuta absoluta Feeding on Tomato Leaves.

Enzymatic Assays and Enzyme Histochemistry ofTuta absoluta Feeding on Tomato Leaves.

Bio-protocol (2018-09-05)
Rim Hamza, José P Beltrán, Luis A Cañas
ABSTRACT

Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed samples to avoid the loss of enzymatic activity. We also describe a protocol for the quantification of trypsin and papain-like enzymes in the larvae of Tuta absoluta at different developmental instars.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride, ≥99% (TLC), suitable for substrate for trypsin
Sigma-Aldrich
Trypsin from bovine pancreas, TPCK Treated, essentially salt-free, lyophilized powder, ≥10,000 BAEE units/mg protein
Sigma-Aldrich
Papain from papaya latex, lyophilized powder, ≥10 units/mg protein
Sigma-Aldrich
L-Cysteine, 97%
Sigma-Aldrich
pGlu-Phe-Leu p-nitroanilide, protease substrate