, 2006; da Silveira et al., 2006, 2007; Appel et al., 2008). Recently, we identified a novel functional isoform of phospholipase-D referred to as LiRecDT7 (L. Vuitika personal communication, 2012). The idea that exogenous brown spider venom phospholipase-D isoforms could be useful reagents

for cell biology studies and can interact with exposed cells arises from the clinical effects triggered following spider bites accidents. Bites evoke a deep and dysregulated inflammatory response related to gangrenous and dermonecrotic loxoscelism (histopathologically characterized Thiazovivin clinical trial as an aseptic coagulative necrosis). The venom also triggers platelet aggregation, causing thrombocytopenia, induces hemolysis and is nephrotoxic (Luciano et al., 2004; da Silva et al., 2004; Swanson and Vetter, 2006). All of these events can be reproduced using purified recombinant brown spider check details phospholipase-D isoforms under laboratory conditions, strengthen the idea that phospholipase-D molecules in the venom play an essential

role in such as activities and could modulate cellular functions (Chaim et al., 2006; da Silveira et al., 2006, 2007; Appel et al., 2008; Kusma et al., 2008; Senff-Ribeiro et al., 2008; Chaves-Moreira et al., 2009, 2011; Chaim et al., 2011). Herein, studying crude L. intermedia venom through a two-dimensional electrophoresis approach using a wide range of pI values Urease (3.0–10.0) in the first dimension, SDS-PAGE for the second dimension, and immunodetection of venom phospholipase-D with a polyclonal antiserum raised against a recombinant form of brown spider venom phospholipase-D (LiRecDT1), we showed that

the venom contains a heterogeneous mixture of proteins (at least 25 spots) ranging in size from 30 kDa to 35 kDa and presenting pI levels ranging from acidic to basic that cross-reacted with antibodies. This result is in agreement with data reported in the literature, which have described crude venom as a mixture of proteins enriched in the low molecular mass range (20–40 kDa) ( Veiga et al., 2000). Our findings also corroborate results in the literature indicating that brown spider venom contains several members of the phospholipase-D family. For instance, eleven intraspecies isoforms of phospholipase-D have been observed in L. laeta venom ( Machado et al., 2005). Finally, our results strengthened the observations of Gremski et al. (2010), who showed that phospholipase-D mRNA accounts for approximately 20.2% of the toxin-encoding transcripts in the L. intermedia venom gland based on transcriptome analysis, and the reported cloning of seven phospholipase-D isoforms from the L. intermedia venom gland, as noted above.