Fig. 2

The composition and activity of venoms spat by and milked from spitting cobras are nearly identical. A Overlaid total ion chromatograms (TICs) and isotope distributions of key toxins from spat (red) and milked (black) venoms with different distributions in the venom gland from N. nigricollis, showing that their peptide toxin composition is identical. B Comparisons of spat and milked venom total ion counts (TICs) from representatives of two independently evolved clades of ‘spitting cobras’ show that they are either identical or have only minor differences in the abundance of peptide toxins. C Non-reduced and reduced SDS-PAGE of spat (S) or post-spit milked (PS) venom suggest that the composition of high-molecular-weight protein toxins is also identical or highly similar. Abbreviations: N. pallida (N. pal.), H. haemachatus (H. hae.), N. nigricollis (N. nig.). D Anticoagulant activity of spat and milked venom on citrated bovine plasma, measured as the sample area under the curve (AUC) minus the mean (m) control AUC, averaged (m (AUC − cAUC)), across four replicates (see Additional file 3). E Cytotoxic activity of spat and milked venom measured via MTT cell viability assay and displayed as the venom concentration that resulted in a 50% reduction in cell viability (IC₅₀) across three replicates (see Additional file 4). F Enzymatic PLA₂ activity of spat and milked venom, measured kinetically and displayed as m(AUC − cAUC), across three replicates (see Additional file 5). Error bars represent the standard error of the mean (SEM) of triplicate readings. Dotted lines in F indicate the activity of positive control venom (Daboia russelii) selected for its high PLA₂ activity to contextualise the findings shown here