Journal: British Journal of Pharmacology

Article Title: From identification to functional characterization of cyriotoxin‐1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

doi: 10.1111/bph.14628

Figure Lengend Snippet: Screening flowchart from crude venom fraction to isolated peptide identification. (a) The collection of 117 venoms was prepared into 7,548 fractions individually added to 384‐well plates (Columns 1 and 2 are 1 μM TTX full block and Columns 3 and 4 are maximal current obtained in extracellular buffer) for testing versus hNaV1.7 channels on the Ionworks Quattro (IWQ, Molecular Devices). The train protocol described as an insert was applied before and after 10‐min incubation of cells with the fraction containing the toxin of interest. Traces of pre‐ and post‐scan are shown in full (b). Also, tonic block obtained on hNaV1.7 channels has been enlarged (c). Source of the screened crude venom is shown in (d). Percentage of inhibition for all tested fractions was plotted. As shown in (e), a threshold was set at 50% block (red line). From the venom library tested, only 8% hits on the hNaV1.7 channels came from scorpion venom, 6% from snake while the majority (86%) was derived from spider venoms (f). (g) Hits were sub‐fractioned and tested against hNaV1.7 and hNaV1.5 channels to identify most promising hits to be further characterized using whole‐cell automated patch‐clamp assays (QPatch)

Article Snippet: Isolation, purification, and de novo amino acid sequencing of CyrTx‐1a A primary high throughput screening of the Smartox venom collection was performed on automated patch‐clamp Ionworks Quattro platform using HEK‐293 cells overexpressing hNa V 1.7 channels (Figure a).

Techniques: Isolation, Blocking Assay, Incubation, Inhibition, Derivative Assay, Patch Clamp

Journal: British Journal of Pharmacology

Article Title: From identification to functional characterization of cyriotoxin‐1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

doi: 10.1111/bph.14628

Figure Lengend Snippet: Flowchart for hNaV1.7 channel hit peptide identification. (a) Percentage of inhibition of peak hNaV1.7 elicited current by application of 0.05 μg of each of the 64 fractions obtained from Cyriopagopus schioedtei spider venom. (b) Each fraction of interest was then separated again to isolate one peptide per well. Sub‐fractions were tested again in our automated patch‐clamp Quattro assay. At this stage, inhibition of hNaV1.7 and hNaV1.5 channels was investigated. (c) Fractionation of the crude venom from C. schioedtei by reversed‐phase chromatography and detection by UV at 214 nm. The fraction containing the peptide of interest is highlighted in green. (d) Cation exchange sub‐fractionation of the primary fraction highlighted in (c). The inset illustrates the MS of m/z 1193.8947 [M + 3H]3+

Article Snippet: Isolation, purification, and de novo amino acid sequencing of CyrTx‐1a A primary high throughput screening of the Smartox venom collection was performed on automated patch‐clamp Ionworks Quattro platform using HEK‐293 cells overexpressing hNa V 1.7 channels (Figure a).

Techniques: Inhibition, Patch Clamp, Fractionation, Reversed-phase Chromatography