Our protocol allows researchers to isolate bioactive signaling molecules used by reptiles in chemical communication. As well as the separation and purification of these compounds for additional testing, or even identification. Using this method, researchers can obtain a source of chemical signals and then extract them for their quantification or bioassay use in isolation.
Demonstrating the procedure today will be Holly Rucker, an honors thesis student from my laboratory. Begin by placing five centimeter squared pieces into a sealable glass container with a hexane compatible lid and adding enough hexane to the container to fully submerge the shed skin pieces before sealing the container. The next day, use clean metal forceps to remove the shed pieces, shaking the pieces as they are captured to retain any remaining hexane within the container.
To determine the extracted lipid mass transfer the extract to a pre weighed, round bottom flask and turn on the vacuum source to the rotary evaporator making sure that the vacuum pressure is sufficient to hold the flask to the neck of the condenser. Open the vent at the end of the condenser, slide the flask onto the neck of the condenser, and close the vent to seal the system. After making sure the flask cannot disconnect from the condenser when the flask is released.
Lower the flask until only the bottom 10%of the flask touches the water in the bath, and start the rotation at medium speed. If the vacuum, speed, condenser flow, and bath temperature are optimal the solvent vapor leaving the flask will condense on the coil and drip into the recovery flask. Evaporate the sample under the vacuum until a bead of liquid with a less than one centimeter diameter is visible in the bottom of the flask.
Then turn off the rotation and vacuum, and raise the flask out of the bath. Hold the flask neck as the vacuum seal is released, and twist the neck to side the flask from the condenser. The bead of liquid in the extract will solidify as the hexane evaporates.
After the bead has dried at room temperature for about five minutes, the lipids will form a translucent white to yellow wax in the flask. Weigh the flask to obtain the final mass and solubilize the lipids in the recorded volume of hexane to yield one milligram or more of lipid per one milliliter of hexane. To prepare a chromatography column, use a wooden dowel rod, longer than the column to position a four-by-four centimeter square of folded fiberglass at the bottom of a glass chromatography column.
Secure the column in a fume hood to a standard ring stand and open the stopcock. Next, pour washed and dried sand into the column until approximately three centimeters of sand rests above the fiberglass. Then, place dark paper under the column and tap it gently.
Now, place a 500 milliliter beaker under the column and slowly wet the sand with approximately 25 milliliters of hexane, closing the stopcock when there is approximately 0.5 centimeters of hexane above the sand. To activate the neutral illumina, pipette deionized water of a volume equal to 6%of the illumina mass in drops throughout the illumina. And cover the flask of activated illumina, vigorously swirling the solution to evenly disperse the charge.
When no visible clumps of illumina remain, add hexane until the illumina is completely covered with about 0.5 centimeters of hexane. Swirl the flask to form a slurry, and place a new 500 milliliter glass beaker beneath the column. Open the stopcock, then swirl the illumina again, before steadily pouring the illumina into a vented funnel held in the reservoir of the column, taking care that the illumina settles evenly within the column and that no large bubbles or cracks form.
The column is formed when the top of the illumina is stable and approximately four centimeters below the neck of the column at the base of the reservoir. Then, use a pipette to rinse the inside of the reservoir with hexane for residual illumina, and gently add a second layer of sand on top of the illumina to approximately one centimeter below the reservoir. For fractionation of the lipid extract, use a long glass pipette to transfer the lipid sample to the column and slowly pipette the extract to avoid disturbing the sand layer.
Rinse the extract vile with five milliliters of hexane and transfer the wash to the column. When all of the extract had been added, open the stopcock and allow the sample to load into the column. Once the sample is added, the column must remain saturated with solvent.
If the column dries out, the sample will be lost. Place a pre weighed and labeled round bottom flask under the column to collect the first fraction. And use a vented glass funnel to pour 100%percent hexane to the side of the reservoir to avoid disturbing the sand layer.
Then, open the stopcock to begin the elution, closing the stopcock when approximately three milliliters of hexane remain above the sand at the top of the column. As larger animals naturally produce more skin lipids because of their greater total skin surface area, it is important to standardize the extracted lipid mass to the animal's snout vent length. Once standardized to the total shed skin mass, the linear relationship of the extracted skin lipid mass with the mass of the shed skin extracted is completely removed.
Following fractionation, the same standardization approach can be used with the masses of the individual fractions. For example, here each fraction is not contributing equally to the total extracted lipid mass, as the neutral lipids are the dominant set of compounds by mass proportion, compared to each set of more polar lipids. If the identification of compounds is the ultimate goal, use clean glassware and you must avoid plastics to prevent contamination of the samples.
For compound identification, liquid or gas chromatography, coupled with mass spectrometry can be pursued after the samples are collected. Alternatively, bioassays can be conducted to elucidate the sample's bio activity.
