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These are general guidelines, which are not suitable for all venoms, toxins or uses. Venoms are complex mixtures including dozens or even hundreds of different peptides and proteins and different venoms and toxins may have very different solubilities, stabilities and sensitivities. Specific applications may require the use of special procedures.

Please consult the literature and feel free to contact us for additional details.

Visual Inspection

Most spider and scorpion venoms are usually water-clear fluids that dry to a snow-white powdery pellet. Any discoloration or cloudiness may a sign of contamination and we should be contacted for a possible refund or replacement.

Some venoms normally gel if slightly dehydrated and some will become gummy after freeze-thaw or lyophilization and reconstitution. Gels generally dissolve when diluted with saline but gummy venoms may not and actives may have to be extracted.

Scorpion venoms tend to be milky, which can be cleared by centrifugation, and diplocentrid venoms are red if the venoms have not been harvested and stored under anoxic conditions. The red color forms after the venom is milked and reacts with with air.

Storage

Frozen and freeze dried venoms tend to be very stable and may retain much of the activity for years when stored at -20 ^oC or colder. Colder is generally preferred and it is better to keep the temperature very cold and constant, since warming and cooling since this may dehydrate small aliquots of frozen venom from evaporation of water from the venom and condensation elewhere.

Reconstitution of Lyophilized Venoms

Lyophilized venoms are initially dissolved in a small amount of saline to keep the concentration very high. Venoms have their own dispersants, which appear to be more effective at higher concentration.

Addition of 90% of the nominal volume  of water will restore the venom to its initial volume, which can be a useful measure. Venoms tend to have a specific gravity of approximately 1.1 milligrams per microliter and dry to approximately 0.2 milligrams, so we assume that 0.9 milligrams of water was removed during drying.

Distilled or deionized water or a buffered saline is recommended for reconstitution. Any solvents, such as DMSO, tend to reduce yields of protein and active. In general, the use of acidic solutions will also result in lower yields even though purified toxins may be solution in acidic solutions.

Clarification

Reconstituted and thawed venoms are generally diluted and clarified before use. For instance, it is very important to remove insoluble materials before chromatography.

Thawed or reconstituted venom may be diluted 20 times nominal volume and centrifuged for 15-60 minutes in a refrigerated microcentrifuge at its highest speed.  Use the buffered saline that will be used in bioassays or the initial running buffer for chromatography.

Storage of Diluted Venoms

Diluted venoms or concentrates are generally stored frozen or in a refrigerator, though this should be confirmed. For instance, some activities are sensitive to freeze-thaw.

It may be convenient and more consistent results may be obtained by storing smaller aliquots of diluted venom at -20 ^oC or colder.

Screening and Exploratory Bioassays

In vitro bioassays

In general, venoms are not screened at concentrations above 10 microliters per milliliter though the consequences of this might be of interest to some. Higher concentrations tend to produce spurious effects, including disruption or even dissolution of cells.

One option is to run a high-side assay at 1-5 microliters per microliter of perfusate to detect toxins with low abundance or activity with three to four 10X dilutions to detect more potent activities without side effects from interfering or less active components of the venom.

In vivo bioassays

The toxicity of venoms varies considerably with the venom and species being tested. Some venoms can produce a fast reaction and may be lethal at less than

Purification Strategy

It would be impossible to discuss this adequately here since there are so many options and advancements in separation technology.

In brief, classic strategies tend to work well for most toxins. These might include initial crude separations by size exclusion (SEC) and ion exchange (IEC) chromatography followed by higher resolution separations using reverse phase RP-HPLC. In most cases, we do not recommend the use of traditional RP-HPLC (0.1% TFA with an acetonitrile gradient) as a first step since many actives are not stable to 0.1% TFA or may precipitate with other components of the venom during preparation. In general, it is better to use milder starting conditions, reserving RP-HPLC for the final step after compatibility has been confirmed. However, there are exceptions and dilution of venom in 0.1% TFA or acetic acid may be a good way to remove many unwanted components of the venom.

In general, it appears to be better to use higher pH and ionic strength solutions during the initial steps, e.g. 0.1N ammonium acetate, pH 8.5 for SEC or as the starting buffer for cation exchange chromatography (CEX). For instance, CEX with a gradient from 0.1N to 2N ammonium acetate generally provides a decent initial separation of basic proteins and the ammonium acetate will be removed during lyophilization of the fractions. Anion exchange is usually used for acidic proteins.

Safety

In general, venoms have little or no inhalation or oral toxicity though they may be eye or respiratory irritants. Most are only effective by injection though some spiders, e.g. Peucetia viridans and Scytodes, can spit from the venom gland.

Wear suitable protection when working with venoms, including hand, mouth and eye protection. Avoid inhalation of powdered venoms, protect wounds and open sores from contact and avoid the use of sharps or glass containers when possible to reduce the chance of accidental injection.

Wash spills with a strong soapy solution and/or bleach and wash skin with soap and water and scrubbing. Use an eye wash to rinse the eyes thoroughly if there is any contact in or around the eye and get medical attention if the venom is injected by accident.

Venoms have no known environmental hazards so routine waste disposal may be used. Follow all local and organizational standards.