Snake venoms are complex mixtures consisting mainly of toxic proteins that are used during the capture and defense of prey. There is limited knowledge about the protein concentration of snake venom and the biases of different protein determination methods. Here, we assess the ability of the Qubit protein assay, the bicinchoninic acid (BCA) assay, the Bradford assay, and NanoDrop spectrometry (A280 with a mass extinction coefficient of one) to accurately quantify protein concentrations of toxins isolated from venom, including three-finger toxins and phospholipase A2. The Bradford assays severely underestimated three-finger toxin concentrations and NanoDrop spectrometry overestimated phospholipase A2 concentrations, while the BCA assay was the most accurate. Venoms from five major African venomous snake genera were also assessed: coral cobras (Aspidealaps spp.); mambas (Dendroaspis spp.); cobras (Oh well spp.); bush viper (Atheris sp.); add (Bitten spp.); and saw-headed vipers (Ecchi sp.). Protein concentration results were inconsistent between methods. Protein concentrations were found to be lowest for Bitten spp. married and highest for Oh well spp. married and did not vary between species of the same genus. However, Elapidae species generally had venom with significantly higher protein concentrations than Viperidae species. Moreover, there was greater variation between Elapidae species. We also determined wet venom yield and used this to provide a preliminary estimate of the total amount of protein that can be injected during a snake bite. We found snake weight and length affected the wet venom yield for Atheris squamigera but not for Bitis arietans and Echi’s novel. Our results aim to improve our understanding of the physical properties of snake venom.
