2.5. Gelatinase Purification

Hemolymph from 50 late L2 D. melanogaster Nasrallah larvae were pooled in 120 µL of IR, centrifuged 10 min at 500× g and 100 µL of supernatant was collected, diluted 1/5th with 400 µL of Tris 20 mM pH 7.5 before concentration back to 100 µL on a 10k cut-off centrifugal filter (Amicon, Merck Chimie, France). Next, 20 µL of the reconcentrated hemolymph were removed to be used as control. The remaining desalted hemolymph was then mixed with ~500 µL of DEAE-Sepharose, which was previously thoroughly washed in Tris-HCl 20 mM pH 7.5. After 10 min under mild agitation, the beads were centrifuged (500× g, 30 s) and the supernatant stored on ice. The beads were then washed three times in 20 mM Tris-HCl pH 7.5 and resuspended in 400 µL of 50 mM NaCl/Tris-HCl 20 mM pH 7.5 for 10 min under mild agitation. The beads were centrifuged, and the supernatant was removed and stored on ice. This step was repeated with increasing NaCl concentration (250, 500 and 1000 mM in Tris-HCl 20 mM). Each eluate was further concentrated down to 40 µL using 10 kDa cut-off centrifugal filter devices before mixing with non-reducing sample buffer (20 µL). The same protocol was applied with gelatin-sephadex beads (Cytiva, Saint-Germain-en-Laye, France). Half of each concentrated elution volume was loaded onto a 12.5% SDS-PAGE gel, and the other half was loaded onto a gelatin-containing 12.5% SDS-PAGE. Alternatively, for gelatin beads after hemolymph binding and washing steps, instead of salt desorption, the beads were directly treated with 50 µL of non-reducing sample buffer to ensure that all proteins were extracted. The same binding protocol was used with benzamidine Sepharose 4FF beads (Cytiva, Saint-Germain-en-Laye, France), but after binding and washing, the elution was performed with 20 mM of 4-benzamidine in Tris 20 mM, pH 7.5 and then the beads were mixed with non-reducing sample buffer to extract the still bound proteins. All experiments were performed at least twice.