Name: extraction of venom and venom gland microdissections from spiders for proteomic and transcriptomic analyses
Uploaded: 2014-11-03T14:00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses at JoVE.com

I thank the following individuals for their assistance in the development of this protocol: Chuck Kristensen, Greta Binford, Alex K. Lancaster, Konrad Zinsmaier, and Mays Imad. Mass spectrometry and proteomics data were acquired by the Arizona Proteomics Consortium supported by NIEHS grant ES06694 to the SWEHSC, NIH/NCI grant CA023074 to the AZCC and by the BIO5 Institute of the University of Arizona. Funding for this work was provided from the National Institutes of Health (National Institute of General Medicine) from grants 1F32GM83661-01 and 1R15GM097714-01 to Jessica E. Garb.

1. Preparation of Electro-stimulator Apparatus
<ol>
	<li>Connect electro-stimulator power cord to outlet and attach external foot pedal to the external signal input. 
	Note: Electrical wires must be properly insulated and exposed metal delivering current should not be touched. Wear latex or nitrile gloves for protection. Electro-stimulator power switch should be in OFF position when attaching power cord and attaching wires.</li>
	<li>Connect positive electrode to red output terminal on electro-stimulator (when polarity switch is in normal mode) and connect the negative electrode to the black output terminal. Note: Each electrode wire should terminate in an alligator clip.</li>
	<li>Set stimulator to the following recommended initial settings: voltage=7 V, Frequency=1 pulses per second, delay=0 milliseconds, duration=200 milliseconds, twin pulses switch=regular, and mode switch=off.</li>
	<li>Test output of electrodes by attaching alligator clips to voltmeter positive and negative test leads. Turn on stimulator power switch and deliver current with foot pedal.</li>
</ol>
2. Preparation of Immobilizing Forceps and Other Materials
<ol>
	<li>Dip one prong of featherweight forceps into liquid plastic coating and wait four hours to let coating dry. Tightly wrap 15 mm of the tip of the opposing prong with a single layer of cotton sewing thread, and secure thread by tying the end in a knot. 
	Note: The thread is used to absorb an applied saline solution that promotes electrical conductivity, while the plastic coating provides insulation to retard current.</li>
	<li>Cut tip of blunt hypodermic needle with sharp scissors and smooth the opening with sand paper. Attach the needle to a vacuum waste trap (e.g., vacuum filter flask) via tubing. 
	Note: &#160;The needle will suction away water and regurgitate, and will serve to complete the circuit created by the stimulator electrodes. The needle opening must be small enough to not harm the spider, yet large enough to vacuum solution without clogging (e.g., 25 G x 1 &#189; in gauge, see Materials List).</li>
	<li>Position featherweight forceps horizontally in a tightly closed position using a vinyl-covered two-pronged clamp secured to a magnetic base or fixed stand apparatus under a dissecting microscope&#8217;s field of view, with plastic coated prong of forceps on top and thread coated prong on bottom. 
	Note: The two-pronged clamp is maintained in a stationary position to the magnetic base by a clamp holder (see Materials List). A rubber band may be tightly secured around prongs of the featherweight forceps, next to the clamp holder, to add additional pressure to keep the featherweight forceps in a closed position around the spider once introduced.</li>
	<li>Attach positive electrode alligator clip to forceps&#8217; bottom prong upstream of thread and attach negative alligator clip to blunt metal vacuum needle.&#160;</li>
	<li>Test circuit current with voltmeter by placing positive lead on forceps prong between thread and alligator clip and placing negative lead on blunt vacuum needle. Press foot pedal to ensure sufficient current is detected and remove leads if sufficient current is detected.</li>
	<li>Prepare several microcapillary tubes for venom collection. Hold a single microcapillary tube at one end with a gloved hand and at the other end with sterile metal forceps, placing the forceps-held end horizontally over a Bunsen burner flame. Pull on the microcapillary with the metal forceps away from the flame to create an elongated tip, while holding the other end in a stationary position. 
	Note: wear protective eyewear as microcapillaries can easily fracture.</li>
	<li>Rest prepared microcapillaries on a mounting putty strip in a petri dish. Examine microcapillary ends under a dissecting microscope and create a small, beveled opening at the pulled end with sterilized metal forceps.</li>
	<li>Label variable number of sterile 0.5 ml tubes. Add sterile, deionized water to sterile tubes (e.g., 5 &#956;l of autoclaved deionized water). Close tubes and place tubes on ice.</li>
	<li>Prepare saline solution in a beaker and fill a second beaker with autoclaved water.</li>
</ol>
3. Immobilization of Spider in Forceps
<ol>
	<li>Turn on CO2 tank and transfer spider from closed plastic collecting vial (see Materials List) into CO2 chamber using long metal forceps. 
	Note: black widow spiders have hazardous venom; always wear gloves during this protocol.</li>
	<li>Keep spider in chamber for 5-10 min or until anesthetized and no longer moving when gently prodded with long forceps. Use transfer pipette to wet thread on forceps with saline solution. 
	Note: For guidelines on CO2 anesthesia parameters for black widows, refer to Spagna and Moore19.</li>
	<li>Retrieve anesthetized spider from CO2 chamber by picking it up by its anterior legs using blunt dissecting forceps and gloved hands. Move anesthetized spider to featherweight forceps apparatus. Separate prongs of closed featherweight forceps and place the spider&#8217;s cephalothorax in between the prongs and allow the prongs to close shut to ensure spider is held tightly in place but is not being crushed. Ensure that the spider is placed carapace dorsum side downwards (see Foelix20 for guide to spider anatomy), resting on thread coated prong, and the carapace&#8217;s ventral side facing the plastic coating, while the positive electrode remains attached to bottom prong. Work quickly when handling spider.</li>
	<li>Adjust dissecting microscope magnification, focus, and light source until the spider&#8217;s chelicerae and fangs are clearly visible.</li>
</ol>
4. Venom Collection
<ol>
	<li>Turn on vacuum and spray spider chelicerae with water solution using a second blunt tipped syringe needle. Suction away water with vacuum needle to remove impurities.</li>
	<li>Touch the vacuum needle with attached negative electrode to spider rostrum and deliver pulse with foot pedal one or more times. Vacuum away regurgitate if necessary. 
	Note: spider rostrum is inside mouth posterior to chelicerae on dorsal surface between chelicerae and endites (see Foelix20 for detailed spider anatomy). Note: With each pulse, the spider&#8217;s legs contract, and venom will be visible as clear droplets emerging from fangs.</li>
	<li>Collect venom droplets with microcapillary tip and transfer capillary tip into 0.5 ml tube with water or buffer solution (solution will be sucked into capillary). 
	Note: avoid puncturing spider with capillary which may lead to hemolymph contamination and death. Also avoid contamination of capillary with silk and glue from spinnerets.</li>
	<li>Attach transfer syringe with tube adapter to microcapillary (at end opposite to collection tip) and dispense venom solution back into tube. Put tube on ice. 
	Note: Dispose of waste capillaries in a nearby sharps container.</li>
	<li>Place open plastic collecting vial, along with its cap, close to the spider. Carefully grasp the spider legs with blunt dissecting forceps held in one hand and carefully open featherweight forceps prongs with other hand, sliding spider into collecting vial with blunt forceps and quickly close cap. Continue with next spider and store venom-containing tubes in -80 &#176;C freezer when finished. 
	Note: continue to exercise caution when moving the spider from the forceps back into the vial. Make sure the collecting vial and cap is nearby to the spider for its rapid transfer and wear gloves.</li>
</ol>
5. Venom Gland Dissections
<ol>
	<li>Two-to-three days after venom collection, anesthetize spider in CO2 chamber (see step 3.1). Fill liquid nitrogen carrier and place next to dissecting microscope. 
	Note: when working with liquid nitrogen, use eye protection and cryogenic gloves.</li>
	<li>Clean dissection area and dissecting forceps with solution that eliminates RNase and DNA contamination. Fill a small petri dish placed under dissecting microscope with dissecting buffer (e.g., 1x saline-sodium citrate (SSC) buffer).&#160;</li>
	<li>Transfer spider from CO2 chamber to petri dish and use forceps to quickly separate the cephalothorax from the abdomen.</li>
	<li>Hold the cephalothorax under the dissecting microscope with forceps and position the chelicerae into field of view. Use the sharp ends of a second pair of forceps to cut cuticle joining the carapace to the lateral aspects of the chelicerae. Laterally grasp the chelicerae using the second pair of forceps, and gently tug back and forth until venom glands are pulled out.&#160;</li>
	<li>Separate glands from the chelicerae (or leave attached to chelicerae if desired) and transfer to a cyrosafe 0.5 ml tube. Place closed tube in liquid nitrogen. 
	Note: each dissection should take no more than 15 min.</li>
	<li>Repeat dissection with additional spiders until finished. Transfer tubes from liquid nitrogen into -80 &#176;C freezer.</li>
</ol>

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M., Bulaj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+and+cardioprotective+conopeptides:+An+emerging+class+of+drug+leads.">Neuroprotective and cardioprotective conopeptides: An emerging class of drug leads.</a> Curr. Opin. Drug Disc. 12 (2), 231-239 (2009).</li><li>Casewell, N. R., W&#252;ster, W., Vonk, F. J., Harrison, R. A., Fry, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+cocktails:+the+evolutionary+novelty+of+venoms.">Complex cocktails: the evolutionary novelty of venoms.</a> Trends Ecol. Evol. 28 (4), 219-229 (2013).</li><li>Vetter, R. S., Isbister, G. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latrophilin+1+and+its+endogenous+ligand+Lasso/Teneurin-2+form+a+high-affinity+transsynaptic+receptor+pair+with+signaling+capabilities.">Latrophilin 1 and its endogenous ligand Lasso/Teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.</a> PNAS. 108 (29), 12113-12118 (2011).</li><li>Williams, J. A., Day, M., Heavner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ziconotide:+an+update+and+review.">Ziconotide: an update and review.</a> Expert Opin. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safe+immobilization+by+CO2+of+Latrodectus+hesperus+(Arachnida:+Theridiidae).">Safe immobilization by CO2 of Latrodectus hesperus (Arachnida: Theridiidae).</a> Pan-Pac. Entomo. l. 74 (4), 210-213 (1998).</li><li>Foelix, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Spiders. , (2010).</li><li>Haney, R. A., Ayoub, N. A., Clarke, T. H., Hayashi, C. Y., Garb, J. 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The author has nothing to disclose and no competing financial interests.

Venoms represent an important source of physiologically reactive proteins, peptides and other molecules with applications for drug discovery, as well as for fundamental aspects of cellular and ecological research1&#8211;3. However, the collection of venom, particularly from dangerous or small animals, is a challenging task. This protocol demonstrates how venom and venom glands can be collected from black widow spiders, and confirms the success of this approach via a combination of MuDPIT analysis of the venom and a protein database derived from cDNAs cloned from venom glands21. While this protocol works well for black widows and medium sized spiders, other venom collection techniques have been employed for larger mygalomorph (tarantula-like) spiders, such as direct aspiration of venom from fangs into glass pipettes e.g., 24. This latter approach, however, will not work well for smaller-sized spiders that are not aggressive.&#160;

One especially critical aspect of the venom collection protocol described here is the initial phases of preparation and optimization of the collection process so that it becomes more routine, consistent and quicker. The protocol is initially challenging to master, but with repeated trials, it becomes easier and faster. Caution is also urged at all critical phases involving the handling of hazardous spiders, the use of electrical current, fine-point glass micro capillaries, and syringe needles. It is important to wear appropriate personal protective equipment such as nitrile gloves, a lab coat, long pants and closed shoes, as well as eyewear when shaping micro capillaries.&#160;

Another challenging aspect to venom collection is the small amount of venom produced by any one spider, particularly Latrodectus species, from which the amounts collected may be limited to 1-2 microliters per individual at best. Obtaining sufficient venom for downstream applications, such as protein gels or functional assays may require the combination of venom from multiple individuals into one tube. In such cases, venom should only be combined from individuals of the same sex, ontogenetic stage, and population given the recognition of intersexual, developmental and geographic variability in some venoms25,26. Spiders may also exhibit considerable variation in the amount of venom produced among individuals, where lesser amounts may reflect the recent depletion of the gland. Thus it may be advisable to collect venom several days after their last feeding. If little venom is released, excessive current should not applied to the spider, which may cause the cuticle to rupture, leading to contamination of the venom with hemolymph or death.&#160;

Contamination of venom samples with spider silk or human sources should also be avoided through the use of sterile or clean equipment. Despite these challenges, collection of pure venom, leaving the spider alive, is preferable to methods that obtain venom from gland homogenates (which do not separate venom components from other cellular proteins) and kill the spider. It is also critical to ensure that samples are quickly frozen to prevent protein degradation.

The extraction of venom promotes subsequent venom production, thereby stimulating venom gene expression in the venom gland. Thus, because this protocol allows for spiders to survive venom depletion, their glands may be dissected several days later (killing the spider) at a point where venom gene expression is expected to be sufficient for genetic studies, such as transcript cloning10,11. Several important precautions must also be taken in venom gland dissections. Emphasis should be placed on using lab equipment and reagents that are free of RNases that degrade RNA. Thus it is recommended to wipe forceps and other non-disposable equipment and surfaces with solutions that eliminate RNase and DNA contamination. The dissections should be performed as quickly as possible and directly frozen to further ensure RNA integrity of the tissue. Finally, dissections should only be performed on anesthetized spiders, after their cephalothorax and abdomen are quickly separated.

In conclusion, this article provides a verified protocol to obtain spider venom and venom glands. Venom and venom glands allow for the isolation and characterization of their protein and peptide components using proteomic and transcriptomic approaches. In addition, venom samples may represent the starting point of functional assays, which determine the biomedical and pharmacological potential of their constituent molecules. Nearly all spiders produce venom, and the wide diversity of venom components synthesized by individual species suggests a vast diversity of venom molecules are yet to be discovered13. Accordingly, this protocol provides tools to investigate the rich source of biologically active molecules present in spider venoms.

Venoms are animal secretions predominantly injected into another animal for the purposes of predation or defense, and also have important biological applications with biomedical relevance1&#8211;3. Only certain animals synthesize venom, but its production is taxonomically widespread across invertebrates (e.g., cnidarians, cone snails, scorpions, and spiders) and vertebrates (e.g., snakes, some fish and mammals), because it has independently evolved multiple times1,4. Biochemical characterization of venoms typically show they are composed of a wide variety of proteins and peptides, which largely act on the circulatory and nervous systems of injected animals to rapidly deliver constituent toxins1. A small fraction of venomous animals may pose a threat to humans, particularly species with synanthropic distributions5. The study of venom composition and functional activities has played an important role in the functional characterization of vertebrate cellular components (particularly neuronal ion channels)6 and in elucidating fundamental cellular processes (e.g. neurosecretion)7. Moreover, select venom peptides have useful properties in biomedical contexts, including their uses as treatments for cancer and pain8,9, and are mined for candidate drug leads and antivenom development. Venoms also play prominent roles in ecological and evolutionary research1,4,10,11, thus the collection of these hazardous secretions has many utilities.

There are &#62;40,000 described species of spider (Order Araneae), and all but one of the more than 100 spider families possess paired venom glands that terminate in fangs12. The high species diversity of spiders suggests that they represent the largest clade of venomous organisms. However, biochemical characterization of spider venoms has largely concentrated on a small number of species most often associated with human envenomation. Recent proteomic and transcriptomic studies of spider venoms indicate they typically contain many unique proteins and peptides2,13,14. Advances in high-throughput cDNA sequencing and mass spectrometry peptide fingerprinting has greatly facilitated the discovery of these venom proteins15. Nevertheless, such work begins with the collection of sufficient venom and/or venom glands from the spiders, and detailed documentation for such techniques are few16.

This paper presents a protocol for the collection of venom and venom glands from black widow spiders, which can be applied to similarly sized spiders. The collection of venom separately from the glands enables identification of proteins that are secreted into the venom as opposed to proteins performing other cellular functions. Black widows and other Latrodectus species are widely recognized as being among the most hazardous spiders due to their highly neurotoxic venom, which causes severe pain in humans, which is sometimes accompanied by profuse sweating, muscle contractions, hypertension, difficulty breathing and patchy paralysis5. The venom collection protocol presented here uses electro-stimulation to deliver electrical current to anesthetized spiders to elicit muscular contractions and release of venom. Venom droplets are quickly collected with micro-capillaries and dispensed into tubes for freezer storage. Because the protocol involves hazardous procedures, it should only be performed by well-trained individuals and caution is urged at key steps. The collected venom has numerous uses, such as characterization and isolation of constituent molecules2, for physiological experiments or functional assays18, and to stimulate venom gene expression11. The protocol concludes with a description of venom gland dissection and preservation useful for the cloning of venom-specific genes, the expression of which has been shown to occur 2-3 days following venom depletion in different spiders10,11.

<table border="0" cellpadding="0" cellspacing="0" width="1090">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Nerve and muscle stimulator (electro-stimulator)&#160;</td>
			<td style="width:153px;">Grass Technologies</td>
			<td style="width:107px;">SD9</td>
			<td style="width:435px;">http://www.grasstechnologies.com/products/stimulators/stimsd9.html</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Voltmeter</td>
			<td style="width:153px;">RadioShack</td>
			<td>22-223</td>
			<td>any generic voltmeter/multimeter can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pointed Featherweight Forceps</td>
			<td style="width:153px;">Bioquip</td>
			<td>4748</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasti Dip</td>
			<td style="width:153px;">Performix</td>
			<td></td>
			<td>Available at Ace Hardware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 G X 1 1/2in Precision Glide hypodermic syringe needle</td>
			<td style="width:153px;">BD Medical&#160;</td>
			<td>305190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum filter flask 1L</td>
			<td style="width:153px;">Nalgene</td>
			<td>DS4101-1000</td>
			<td>smaller flask sizes may also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buchner Two Piece funnel, 90 mm</td>
			<td style="width:153px;">Nalgene</td>
			<td style="width:107px;">4280-0900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 microlitter Capillary Bores (Micro capillaries)</td>
			<td style="width:153px;">VWR</td>
			<td>53508-375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting putty strip&#160;</td>
			<td style="width:153px;">Loctite</td>
			<td></td>
			<td>Available at Ace Hardware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand* General-Purpose Extra-Long Forceps Length: 11-13/16 in.</td>
			<td style="width:153px;">Fisher</td>
			<td>10-316C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SSC Buffer, 20X (pH 7.0), Molecular Grade</td>
			<td style="width:153px;">Promega</td>
			<td>V4261</td>
			<td>can be made from stock chemicals (150 mM NaCl, 15 mM sodium citrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf safe-lock tubes 0.5 mL tubes (cryogenic safe)</td>
			<td style="width:153px;">Eppendorf</td>
			<td>22363611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene Dewar, 1L, HDPE, Liquid nitrogen benchtop flask</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td>4150-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rnase Away</td>
			<td style="width:153px;">VWR</td>
			<td>53225-514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Fine Tweezers (dissecting forceps)</td>
			<td>EMS</td>
			<td>78310-0</td>
			<td>similar high-quality fine point forceps can be substituted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Foot switch/pedal</td>
			<td style="width:153px;">Linemaster Switch Corp.</td>
			<td style="width:107px;">491-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Two-pronged extension clamp, with vinyl covered sleeves, 8.5 inches</td>
			<td style="width:153px;">VWR</td>
			<td style="width:107px;">21570-007</td>
			<td>similar models could be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Clamp Holder</td>
			<td style="width:153px;">VWR</td>
			<td style="width:107px;">89084-746</td>
			<td>similar models could be substituted, must accommodate diameter of extension clamp rod</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Magnetic base (holds extension clamp via clamp holder)</td>
			<td style="width:153px;">VWR</td>
			<td style="width:107px;">300042-270</td>
			<td>similar apparatus able to securely hold extension clamp in fixed position may be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Plastic Collecting Vials (large/40 dram)</td>
			<td style="width:153px;">Bioquip</td>
			<td style="width:107px;">8940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Cotton sewing thread</td>
			<td style="width:153px;">Threadart.com</td>
			<td style="width:107px;">&#160;THRCOT9</td>
			<td>similar product could be substituted</td>
		</tr>
	</tbody>
</table>

extraction of venom and venom gland microdissections from spiders for proteomic and transcriptomic analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

The collection of venom and venom gland dissections are frequently performed to characterize venom proteins and peptides at the sequence level10,11,15. Collected venom may also be used in physiological assays to determine their functional activities18. The collection of venom will stimulate venom gland gene expression, thereby facilitating the cloning of specific toxin transcripts via RT-PCR10,11. Identification of venom proteins can also be accomplished in a high-throughput manner by integrating standard mass spectrometry techniques with sequence databases generated from venom gland cDNA libraries15,21. An example of such work, starting from venom and glands collected using the protocol detailed above, demonstrating its effectiveness, is illustrated in Figure 1.
Venom collected from L. hesperus adult females was digested with trypsin and subjected to an in solution MuDPIT analysis, which links HPLC (high performance liquid chromatography) to tandem mass spectrometry (MS/MS). Here the MuDPIT analysis was performed by the University of Arizona Proteomics Consortium. Masses of detected peptides and their dissociated fragments (daughter ions, inferred from spectra) were compared to peptide sequences theoretically predicted from translations of cDNA sequences obtained from a gene expression library constructed from L. hesperus venom glands (glands were acquired by the dissection protocol described above)21. In this experiment, 36 proteins were detected from all collected spectra, at the 99.9% probability threshold, and contained at least one detected peptide. One of the detected proteins is supported by 43 total spectra (exemplar spectra shown in Figure 1A), corresponding to three exclusive peptides, covering 35% of the protein sequence (Figure 1B). The detected protein (translated from a collected cDNA) has a top BLASTp hit to latrodectin from L. tredecimguttatus (GenBank Accession P49125.1), with an e-score of 2e-46, and shares 80% identity at the amino acid sequence level with the protein detected in this experiment. Latrodectin, which is also known as alpha-latrotoxin low molecular weight protein, is a recognized venom component of black widow spiders22,23, verifying the effectiveness of the presented protocol. Some proteins identified from the venom correspond to sequences for which no BLAST hit is found. It is unclear whether such a result is due to the limited genomic resources available for spiders as well as the limited functional information for their proteins, or because the unknown protein represents a venom contaminant. Nevertheless, gene or protein expression analyses examining the abundance of such a novel protein in venom glands relative to other tissues should reveal whether it is a genuine venom component.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51618/51618fig1highres.jpg" width="600" /> 
Figure 1: Latrodectin peptide identified from black widow venom using mass spectrometry. (A) Representative spectra (one of 43) detected via MS/MS portion of MuDPIT analysis of L. hesperus venom which was assigned to predicted translation of a collected cDNA sequence shown in part B. Spectra shows mass to charge ratio (m/z) of detected daughter ions on the horizontal axis produced by fragmenting parent peptide (CGEEDFGEEIVK), with letters at top indicating the peptide sequence based on daughter ion masses. (B) Protein sequence to which spectra in part A was assigned, showing in red, bold and underlined text the corresponding sequence from the spectra shown in part A, additional red text (not bold) represents another peptide in this protein detected with other collected spectra. The protein sequence is translated from a cDNA sequence obtained from dissected venom glands.&#160;

Watch this Scientific Journal Video about Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses at JoVE.com

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

extraction-venom-venom-gland-microdissections-from-spiders-for

Research

JoVE Journal

Biology

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

Rearing of Brazilian Yellow Scorpion for Venom Extraction

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.

Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow ...

Housing, Captive Maintenance, and Feeding of Brazilian Yellow Scorpions for Venom Extraction

Begin by preparing the enclosures for housing the scorpions. To smoothen the walls of the polypropylene container acting as an enclosure, place 10 centimeters of self adhesive plastic tape over its internal surface, as well as on the upper corners of the container. Then, line the floor of the polypropylene container with craft paper fixed with masking tape on the edges on all sides.Arrange four cardboard trays as vertical substrates on one side of the container with a rigid cardboard sheet between each tray. Place a shallow polypropylene tray containing soaked cotton as a water source on the opposite side of the enclosure. Place the scorpions in the prepared enclosures and house the containers in the room maintained at 24 degrees Celsius.For complete maintenance, first remove the scorpions from the water tray. Then, using long forceps remove the water tray from the enclosure. Place the scorpions in the clean enclosure.Remove all the scorpions left on the enclosure's floor and place them in the clean enclosure. Then remove the remaining live prey from the enclosure. Remove all the disposable substrates from the enclosure and properly discard them before sanitizing the reusable items such as water trays and containers with 0.5%hypochlorite solution.To begin, place the scorpions in communal polypropylene containers as enclosures. Calculate the amount of feed according to the energy intake of the scorpions. Then, count the number of cockroaches as prey items.Then, apply calcium carbonate with alcohol around the top of the enclosure to prevent cockroaches from escaping the enclosure. Place the cockroaches inside the enclosure for two days.