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Purification

 

Use Suggestions for Arthropod Venoms

Obviously, no single technique is suitable for all toxins, venoms or projects. There may be hundreds of thousands or millions of different toxins, venoms with a wide range of properties and innumerable possible applications.

 

In general, spider venoms are very soluble in neutral to basic (pH 7-10) buffers with an ionic concentration of at least 0.1N. We generally see significant amounts of precipitate in dilutions in acidic and hypotonic solutions.

 

Lyophilized venom reconstitution and dilution

Many venoms contain high concentrations of solubilizers or surfactants that may be more effect at higher concentrations.

We usually add the nominal volume of distilled water or a buffered saline to the lyophilized venom and agitate this with the pipette tip. Alternately, add 0.9 times the nominal volume to approximate the initial concentration of the venom. Venoms typically lyophilized to approximately 0.2 mg/uL and have a specific gravity of approximately 1.1, so addition of 0.9 uL water / uL lyophilized venom should restore the initial concentration of the venom.

 

Reconstitution for reverse phase chromatography in TFA

Add 5 uL distilled water to 5 uL lyophilized venom

Agitate with pipette tip

Dilute with 45 uL 0.1% TFA

Centrifuge in a microcentrifuge for 15-30 minutes at full speed

 

Screening concentrations

Concentrations of venom above 5-10 microliters per milliliter tend to produce spurious effects and high concentrations may even dissolve membranes and cells while toxins with low activity or low concentrations in the venom may 

 

SDS Electrophoresis

I am not sure of the reason, but have found that it usually helps to increase the concentration of SDS to 10%. Otherwise, some of the venom may precipitate.

 

Native Electrophoresis

Routine methods usually work for venoms though remember to run anionic as well as cationic gels since many toxins are highly basic. Gradient gels containing 5-25% acrylamide are useful since toxin range in size from less than 1kD to more than 100,000 kD.

Terms

Nominal volume refers to the initial quantity of neat venom, as it is harvested from the animals. Spider Pharm usually aliquots venom by neat volume, undiluted as it is collected from the fangs, so nominal volume is usually the quantity listed on the vial from Spider Pharm.

Lyophilized is equivalent to freeze-dried. We generally dry the venom under vacuum, keeping the venom at -20oC or colder during the drying process.

Typical concentrations:

10% w/v protein

20% w/v solids