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Using venoms
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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. |
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