Amicon ultra centrifugal filter

The following were used:AmiconUltra centrifugal filters (#UFC200324), Pierce streptavidin magnetic beads (Thermo Fisher Scientific, #88816), Bolt Antioxidant (Thermo Fisher Scientific, #BT0005), Imperial protein stain (Thermo Fisher Scientific, #24615), Pierce Top 12 Abundant Protein Depletion Spin Columns (Thermo Fisher Scientific, #85165), AminoLink Plus Immobilization Kit (ThermoFisher Scientific, #44890), NuPAGE Bis-Tris and Tris-Glycine gels (Thermo Fisher Scientific), human adiponectin (R&D Systems, #1065-AP-050), human C1q (Abcam, #ab96363), human collagens type I and II (Abnova, #P4915 and #P4916), and human collagen typeVIII a1 anda2NC1domains (Antibodies Online, #ABIN1079239 and #ABIN1098982).

Purified His₆-TEV-RBPMS-A was exchanged to 500 mM KCl AUC buffer using a Zeba spin desalting column (7K MWCO, Thermo Fisher Scientific). Alexa Fluor 647 C2 maleimide (Thermo Fisher Scientific) was added to 10× molar excess and incubated overnight at 4°C in the dark. The reaction was quenched with excess β-mercaptoethanol and buffer exchanged to QA buffer. Using an Amicon Ultra centrifugal filter (10K MWCO; Thermo Fisher Scientific), labelled protein was repetitively concentrated until a 100 000× dilution was achieved. The amount of free fluorophore in the mixture was estimated by SDS–PAGE (Supplementary Figure S4A).

The initial fractionation of the crude venom was performed by the Amicon Ultra Centrifugal Filter (#UFC801008; Thermo Fisher Scientific, Suwannee, GA). This procedure consisted of separating crude venom by molecular mass using molecular filters, generating three main fractions named: F1 (low weight, less than 3 kDa), F2 (intermediate weight, between 3 and 10 kDa) and F3 (high weight, more than 10 kDa). From these, experiments were conducted to select the most significant fraction, considering the antineoplastic effects; Then, F1 and F2 were chosen and F3 was eliminated. Further purification of F1 and F2 was carried out: reversed phase HPLC was performed using a Shimadzu VP-ODS column, 0.1% trifluoroacetic acid (TFA) as mobile phase and 90% acetonitrile 0.1% TFA as eluent. More purified components were obtained, named subfractions 1—12 (SF1—SF12).

Two samples of lyophilized crude venom were extracted by electrical stimulation of numerous adult spiders of both sexes (Sisgen A23162A). The crude PnV, fractions, and subfractions were stored at −80°C and dissolved in a sterile culture medium immediately prior to use. The composition and reproducibility of the venom lots were verified by high-performance liquid chromatography (HPLC). The initial fractionation of the crude venom was performed using the Amicon ultra centrifugal filter (#UFC801008; Thermo Fisher Scientific, Suwannee, GA, United States). This procedure consisted of the separation of the crude venom by molecular mass using molecular filters, generating 3 main fractions that were denominated: F1 (LW = low weight, less than 3 kDa), F2 (IW = intermediate weight, between 3 kDa and 10 kDa), and F3 (HW = high weight, more than 10 kDa). From these, experiments were conducted to select the more significant fraction, considering the effects presented; then, F1 and F2 were chosen, and F3 was eliminated. A new purification of F1 and F2 together, using HPLC, was carried out, obtaining new components, which were denominated subfractions 1 to 11 (SF 1–SF 11). Reversed phase HPLC was performed using a Shimadzu VP-ODS column, with 0.1% trifluoroacetic acid (TFA) as the mobile phase and 90% acetonitrile 0.1% TFA as the eluent.