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. 1999 Jan 19;96(2):754–759. doi: 10.1073/pnas.96.2.754

Figure 2.

Figure 2

(A) Cation exchange chromatography of crude P. australis venom. Fifty milligrams of venom was applied to a Bio-Rad S-50 cation exchange matrix and eluted with a KCl gradient. Shown is the column profile at 280 nm. Inhibitory activity eluted near the end of the gradient. The indicated fractions were pooled for RP-HPLC. (B) Reversed-phase chromatography of pooled cation exchange fractions. Cation exchange fractions were pooled from 100 mg of raw venom and applied to a C4 reversed-phase column. Components were differen tially eluted with a water/acetonitrile gradient (0.1% TFA throughout). (Upper) The column profile at 280 nm. The 24-kDa component, PsTx, eluted at 32–34 min as a broad peak as indicated by the arrow. One-minute fractions were collected and dried. After being redissolved in 1 ml of water, a portion of each fraction was diluted 1:30 into control buffer containing BSA and applied to the extracellular face of a membrane patch expressing the rat olfactory channel. (Lower) the fraction current inhibition at +80 mV (upward, black) and at −80 mV (downward, gray). (C) SDS/PAGE analysis of a final RP-HPLC purified PsTx preparation. Shown is a SDS–15% polyacrylamide gel containing 1 μg of PsTx after staining with Coomassie brilliant blue.