The overall goal of this procedure is to extract venom and dissect venom glands from spiders for transcriptomic and proteomic studies, this is accomplished by first preparing the equipment and reagents needed to collect venom by electrical stimulation. The second step is to immobilize an anesthetize spider with forceps under a dissecting microscope. Next, the spider is pulsed with current and the released venom is collected with a micro capillary tube.
The final step is to dissect the spider's venom glands two to three days later. Ultimately, mass spectrometry or other proteomic analyses of the venom is used to show the sequences of constituent venom proteins. Visual demonstration of this method is critical as the venom collection steps are difficult to learn because they involve a sequence of coordinated actions that must be performed quickly, yet carefully.
To begin, connect an electro stimulator power cord to an outlet and attach the external foot pedal to an external signal input. Next, ensure the polarity switch is in normal mode and connect the positive electrode to the red output terminal on the electro stimulator and connect the negative electrode to the black output terminal. Then set the stimulator to the following recommended initial settings.
Test the electrode output by attaching alligator clips to the positive and negative volt meter Test leads. Then turn on the stimulator power switch and deliver current with the foot pedal. Prepare the featherweight forceps by coating one prong with liquid plastic and wait four hours for the coating to dry after the plastic has dried tightly wrapped 15 millimeters of the AP opposing prong tip with a single layer of cotton sewing thread and secure the thread by tying the end in a knot.
Next, using sharp scissors, cut the tip of a blunt hypodermic needle, small enough not to harm the spider, yet large enough to avoid clogging. Smooth the opening with sandpaper and attach the needle to a vacuum waste trap. Then position the featherweight forceps horizontally in a tightly closed position using a vinyl covered two-pronged clamp secured to a magnetic base under a dissecting microscope's field of view with the plastic coated prong on top and the thread coated prong on the bottom.
Next, attach a positive electrode alligator clip to the bottom prong upstream of the thread, and attach a negative alligator clip to the blunt metal vacuum needle. Then place the positive lead on the forceps prong between the thread and alligator clip and the negative lead on the blunt vacuum needle. To test the volt meter circuit current, press the foot pedal to ensure a sufficient current is detected, and remove the leads while wearing protective eyewear.
Prepare several micro capillary tubes for venom collection. Next, place a mounting putty strip in a Petri dish and rest the micro capillaries on the strip. With a gloved hand hold a single micro capillary tube at one end and hold the other end with sterile metal forceps over a bunsen burner flame and pull the end away from the flame to create an elongated tip while holding the other end in a stationary position, examine the micro capillary ends under a dissecting microscope and create a small beveled opening at the pulled end with sterilized metal forceps.
Close the tubes and place them on ice. To begin spider immobilization, turn on the CO2 chamber gas. Then transfer the spider with long metal forceps from the closed plastic collecting vial into the CO2 chamber.
Keep the spider in the chamber for five to 10 minutes and check that the spider is no longer moving by. Gently prodding it with long forceps. Then use a transfer pipette with saline solution to wet the thread on the forceps.
Next, retrieve the anesthetized spider from the CO2 chamber by picking it up by its anterior legs. Using blunt dissecting forceps and gloved hands, move the anesthetized spider to the featherweight forceps apparatus. Then separate the prongs of the closed featherweight forceps and place the spiders cephalic thax between the prongs.
Allow the prongs to close shut to ensure the spider is held tightly in place but is not being crushed Quickly ensure that the spider is placed carpace dorsum side downwards, resting on the thread coated prong, and the car's ventral side is facing the plastic coating while the positive electrode remains attached to the bottom prong. Next, adjust the dissecting microscope magnification focus and light source until the spiders cery and fangs are clearly visible. To begin venom collection, turn on the vacuum and spray the spider colliery with water.
Using a second blunt tip syringe needle suction away the water with a vacuum needle to remove impurities. Next, touch the vacuum needle with the negative electrode to the spider's rostrum and deliver a pulse with the foot pedal. The spider's legs will contract and venom will be visible as clear droplets emerging from fangs.
Vacuum away regurgitate if necessary. Then collect venom droplets with the micro capillary tip and transfer the capillary tip into a 0.5 milliliter tube with water, which will be sucked into the capillary. Avoid puncturing the spider with the capillary and contamination of the capillary with silk and glue from the spints.
Next, attach the transfer syringe with tube adapter to the micro capillary at the end opposite to the collection tip and dispense the venom solution back into the tube and place the tube on ice store venom containing tubes at minus 80 degrees Celsius. When finished, when finished with venom collection, set an open plastic collecting vial along with its cap close to the spider. For rapid transfer, carefully grasp the spider's legs with blunt dissecting forceps held in one hand and carefully open featherweight forceps with the other hand.
Then slide the spider into the collecting vial with blunt forceps. And quickly close the cap two to three days after venom collection, anesthetize the spider in a CO2 chamber, then fill a liquid nitrogen carrier and place it next to a dissecting microscope. Clean the dissection area and forceps with RNA away.
Then fill a small Petri dish with one x saline sodium citrate, and place it under the dissecting microscope. Next, transfer the spider from the CO2 chamber to the Petri dish and use forceps to quickly separate the cephalic thax from the abdomen. Then hold the cephalic thax under the dissecting microscope with forceps and position the colliery into the field of view.
Use the sharp ends of a second pair of forceps to cut the cuticle joining the carabase to the lateral aspects of the colliery laterally. Grasp the colliery with the second pair of forceps and gently tug back and forth until the venom glands are pulled out. Taking no more than 15 minutes.
Separate the glands from the colliery and transfer them to a cryo safe 0.5 milliliter tube. Place the closed tube in liquid nitrogen, integrating mass spectrometry techniques with sequence databases generated from venom gland CD NA.Libraries enabled the identification of venom proteins. Laro inept peptide was one of 36 proteins identified from black widow venom using mass spectrometry.
Don't forget that working with spiders like black widows can be extremely hazardous, and precautions such as wearing protective gloves should always be taken while performing this procedure.
