Name: A Method for Extracting Pigments from Squid Doryteuthis pealeii (Video) | JoVE Journal
Uploaded: 2016-11-09T15:00:00.000+00:00
Duration: 11 min 3 s
Description: Learn a method for extracting pigments from squid chromatophores using Doryteuthis pealeii. This protocol enables nanoscale analysis of color change mechanisms in cephalopods.

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5 minutes

The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

Invertebrate animal studies conducted herein are not regulated in the United States; therefore the Institutional Animal Care and Use Committee has no authority for review of such protocols. In lieu of these not falling under the jurisdiction of regulation in the United States, the authors hereby state that these studies were conducted with sincere effort towards the ethical use, care, and treatment of these animals, the number of individuals was minimized and these efforts are consistent with the Basel Declaration and the International Council for Laboratory Animal Science (ICLAS) ethics guidelines.
1. Doryteuthis pealeii (D. pealeii) Dissection
<ol>
	<li>Purchase decapitated squid D. pealeii from the Marine Resources Department at the Marine Biological Laboratory at Woods Hole, MA or from any fresh-fish market. Do not use specimens that have been filleted or previously altered in order to apply this procedure. Specifically, the chromatophore layer must still be intact on the specimen.
	<ol>
		<li>If the animals are not used immediately, store them at -20 &#176;C until use. To defrost the specimens, place them in room temperature water for 1 hr prior to dissection.</li>
	</ol>
	</li>
	<li>Using stainless steel straight fine-point dissection scissors, cut along the posterior side of the animal. Begin at the base of the ventral mantle, and end at the fin region.</li>
	<li>Remove all of the internal organs (stomach, gill, ink gland, reproductive organs, systemic heart, and brachial heart) by lifting them with dissecting forceps and severing their connective tissue using dissection scissors. Discard organs in a specimen bag.</li>
	<li>Use dissecting T-pins to pin the mantle (fin side up) in a standard 11-1/2&#34; x 7-1/2&#34; x 1-1/2&#34; aluminum dissection pan containing a flexible dissection pad. Submerge the tissue in 250 ml of seawater that has been filtered through a disposable polystyrene vacuum filtration system containing 0.2 &#181;m pore sizes.</li>
	<li>Carefully remove the epidermal skin layer (opaque color) with dissecting forceps, keeping the chromatophore containing skin layer intact. Once the epidermal layer is removed, discard it in a specimen bag.</li>
	<li>Dissect small (1 cm x 1 cm) sections of the chromatophore layer with forceps and dissection scissors. Collect samples in 1.5 ml micro-centrifuge tubes (fill one-half of each empty tube with the dissected tissue).</li>
	<li>Add 500 &#181;l of filtered seawater to the tissue containing tubes. The samples can be stored overnight at 4 &#176;C.</li>
</ol>
2. Isolating Chromatophore Pigment Granules
<ol>
	<li>Tissue Digestion
	<ol>
		<li>Preparation of Papain/collagenase
		<ol>
			<li>Weigh out 30 mg of collagenase and 5 mg of papain using a 4&#34; x 4&#34; low nitrogen weigh paper. Transfer these into a 50 ml polypropylene centrifuge tube with a screw cap.</li>
			<li>Measure 10 ml of deionized water using a graduated pipette. Add directly to vial containing papain/collagenase. Vortex on the highest setting until the solution is clear.</li>
		</ol>
		</li>
		<li>Removing Extracellular Tissue
		<ol>
			<li>Centrifuge the collected chromatophore tissue from step 1.7 for 5 min at 14,000 x g. Discard the top layer of sea water into a plastic waste bucket.</li>
			<li>Add 500 &#181;l of the papain/collagenase solution to the tissue using a P1000 variable volume single channel micropipette. Vortex each sample for 1-2 min on the highest setting.</li>
			<li>Sonicate the samples for 5 min at a frequency of 40 kHz to dissociate cells from the surrounding tissue.</li>
			<li>Centrifuge for 5 min at 14,000 x g. Discard the supernatant into a plastic waste bucket.</li>
			<li>Centrifuge the collected chromatophore tissue for 5 min at 14,000 x g. Discard the top layer of sea water into a plastic waste bucket. Repeat once again.</li>
			<li>Manually remove any remaining large tissue sections that did not digest in this process with forceps before proceeding to the next step.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Isolating the Pigment Granules
	<ol>
		<li>Preparation of Homogenization Buffer
		<ol>
			<li>Weigh 2.38 g of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES, 100 mM), 0.095 g of magnesium chloride (10 mM), 0.856 g of potassium-aspartate (50 mM), and 0.015 g of dithiothreitol (1 mM). Transfer into a 150 ml polystyrene container with a screw cap. Add one commercial mini tablet protease inhibitor.</li>
			<li>Measure 100 ml of deionized water using a graduated cylinder. Add directly to the container containing the homogenization salts. Vortex until the solution is clear.</li>
		</ol>
		</li>
		<li>Homogenization of Pigment Granules
		<ol>
			<li>Centrifuge each sample from step 2.1 at 14,000 x g for 5 min. Discard the supernatant into a plastic waste bucket. This sample should be void of extracellular tissue.</li>
			<li>Add 500 &#181;l of homogenization buffer to each tube and vortex for 1-2 min each to thoroughly mix the samples. Sonicate the samples for 30 min (~40 kHz) and follow with 5 min of centrifugation at 14,000 x g.</li>
			<li>Discard the supernatant into a plastic waste bucket, and repeat the previous step 2-3 times to ensure the complete digestion of the chromatophore sacculus and protein tethers. The remaining solution should contain a light yellow supernatant and a red-colored pellet containing the isolated pigment granules.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Pigment Extraction
<ol>
	<li>Preparation of Acidic Methanol (HCl-MeOH)
	<ol>
		<li>Carefully add 25 &#181;l of concentrated (36.5-38 % (w/w)) hydrochloric acid (HCl) to 5 ml of methanol (MeOH). Store the HCl-MeOH solution in a screw cap glass sample vial. Swirl gently to uniformly mix the solution. 
		NOTE: The more acidic the solution, the more pigment will be extracted during the first extraction.</li>
	</ol>
	</li>
	<li>Extraction of Pigment
	<ol>
		<li>Centrifuge the pigment granule suspension (from 2.2.2) for 5 min at 14,000 x g and discard the supernatant into a plastic waste bucket.</li>
		<li>Add 500 &#181;l of the HCl-MeOH solution and vortex each sample for ~3-4 min. Sonicate the samples for 10 min (~40 kHz), then centrifuge for 5 min at 14,000 x g.</li>
		<li>Using a transfer pipette, collect the supernatant in a 1 dram screw-top glass vial. The supernatant should be dark red in color.</li>
		<li>Repeat the extraction step 3.2.2 until no further color is extracted (~4-5 washes). 
		NOTE: Each extraction will yield ~1.5 ml of pigment. The remaining colorless pellet is the pigment extracted granules. NOTE: Both colorless granules and extracted pigments can be stored in water at 4 &#176;C until further use.</li>
	</ol>
	</li>
</ol>
4. Characterization throughout Extraction Process
<ol>
	<li>Scanning Electron Microscopy
	<ol>
		<li>Image the granules (unreacted from step 2.2.1 and pigment-extracted from step 3.2.3) using scanning electron microscopy (SEM) to verify the purity of the extraction process.</li>
		<li>To prepare granule samples for imaging, first centrifuge both unreacted granules and pigment extracted granules for 5 min at 14,000 x g, and discard the supernatant into a plastic waste bucket. Re-suspend these in 1 ml of ethanol, and deposit 2-3 drops of the granule suspension onto aluminum SEM stubs using a transfer pipette.</li>
		<li>After drying the samples overnight, sputter coat with gold-platinum coating for 3 min to achieve a 15 nm coating.</li>
		<li>Image using a SEM with a Schottky emitter and a secondary electron detector. Use a 5-7 kV beam voltage and a working distance between 8 and 9 mm to image these materials.</li>
	</ol>
	</li>
	<li>Ultraviolet-visible (UV-vis) Spectrophotometric Measurements
	<ol>
		<li>Monitor the absorbance profiles for all 3 conditions (unreacted granule from 2.2.2, extracted pigment from 3.2.3, and pigment extracted granules from 3.2.4) using a dual beam spectrophotometer from 200-800 nm.</li>
		<li>Collect a blank measurement using deionized water in a 1 ml cuvette for the unreacted granules and the pigment extracted granules. Collect UV-Vis absorption spectra for both samples.</li>
		<li>Collect a blank measurement using the acidic methanol solution in a 1 ml cuvette for the extracted pigment. Collect UV-Vis absorption spectrum for sample.</li>
		<li>Determine maximum wavelength of each sample by identifying the wavelength at which the absorbance peak reaches its maximum relative height.</li>
	</ol>
	</li>
</ol>

<ol><li>Bell, G. R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatophore+radial+muscle+fibers+anchor+in+flexible+squid+skin%5BTitle%5D)+AND+%22Invertebr+Biol%22%5BJournal%5D)">Chromatophore radial muscle fibers anchor in flexible squid skin.</a> Invertebr Biol. 132 (2), 120-132 (2013).</li>
<li>Crookes, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reflectins%3A+The+unusual+proteins+of+squid+reflective+tissues%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Reflectins: The unusual proteins of squid reflective tissues.</a> Science. 303 (5655), 235-238 (2004).</li>
<li>Deravi, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure-function+relationships+of+a+natural+nanoscale+photonic+device+in+cuttlefish+chromatophores%5BTitle%5D)+AND+%22J.+R.+Soc.+Interface%22%5BJournal%5D)">The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.</a> J. R. Soc. Interface. 11 (93), 1-9 (2014).</li>
<li>Mäthger, L. M., Denton, E. J., Marshall, N. J., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+and+behavioural+functions+of+structural+coloration+in+cephalopods%5BTitle%5D)+AND+%22J.+R.+Soc.+Interface%22%5BJournal%5D)">Mechanisms and behavioural functions of structural coloration in cephalopods.</a> J. R. Soc. Interface. 6 (2), 149-163 (2009).</li>
<li>Mäthger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomical+basis+for+camouflaged+polarized+light+communication+in+squid%5BTitle%5D)+AND+%22Biol.+Lett%22%5BJournal%5D)">Anatomical basis for camouflaged polarized light communication in squid.</a> Biol. Lett. 2 (4), 494-496 (2006).</li>
<li>Mäthger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Malleable+skin+coloration+in+cephalopods%3A+selective+reflectance%2C+transmission+and+absorbance+of+light+by+chromatophores+and+iridophores%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores.</a> Cell Tissue Res. 329 (1), 179-186 (2007).</li>
<li>Cloney, R. A., Brocco, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatophore+Organs%2C+Reflector+Cells%2C+Iridocytes+and+Leucophores+in+Cephalopods%5BTitle%5D)+AND+%22Amer.+Zool%22%5BJournal%5D)">Chromatophore Organs, Reflector Cells, Iridocytes and Leucophores in Cephalopods.</a> Amer. Zool. 23 (3), 581-592 (1983).</li>
<li>Hanlon, R. T., Messenger, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adaptive+coloration+in+young+cutttlefish+%28Sepia+officinalis+L%29-+The+morphology+and+development+of+body+patterns+and+their+relation+to+behaviour%5BTitle%5D)+AND+%22Philos+Trans+R+Soc+Lond+B+Biol+Sci%22%5BJournal%5D)">Adaptive coloration in young cutttlefish (Sepia officinalis L)- The morphology and development of body patterns and their relation to behaviour.</a> Philos Trans R Soc Lond B Biol Sci. 320 (1200), 437-487 (1988).</li>
<li>Sutherland, R. L., Mäthger, L. M., Hanlon, R. T., Urbas, A. M., Stone, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cephalopod+coloration+model.+II.+Multiple+layer+skin+effects%5BTitle%5D)+AND+%22J.+Opt.+Soc.+Am%22%5BJournal%5D)">Cephalopod coloration model. II. Multiple layer skin effects.</a> J. Opt. Soc. Am. 25 (8), 2044-2054 (2008).</li>
<li>Florey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+and+function+of+cephalopod+chromatophores%5BTitle%5D)+AND+%22Amer.+Zool%22%5BJournal%5D)">Ultrastructure and function of cephalopod chromatophores.</a> Amer. Zool. 9 (2), 429-442 (1969).</li>
<li>Florey, E., Kriebel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+and+mechanical+responses+of+chromatophore+muscle+fibers+of+squid%2C+Loligo+opalescens%2C+to+nerve+stimulation+and+drugs%5BTitle%5D)+AND+%22Z.+Vergl.+Physiol%22%5BJournal%5D)">Electrical and mechanical responses of chromatophore muscle fibers of squid, Loligo opalescens, to nerve stimulation and drugs.</a> Z. Vergl. Physiol. 65 (1), 98-130 (1969).</li>
<li>Williams, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+Phenoxazone-Based+Pigments+to+the+Structure+and+Function+of+Nanostructured+Granules+in+Squid+Chromatophores%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Contributions of Phenoxazone-Based Pigments to the Structure and Function of Nanostructured Granules in Squid Chromatophores.</a> Langmuir. , (2016).</li>
<li>Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ommochrome+pigmentation+of+the+linea+and+rosa+seasonal+forms+of+Precis+coenia+%28Lepidoptera%3A+Nymphalidae%29%5BTitle%5D)+AND+%22Arch.+Insect.+Biochem.+Physiol%22%5BJournal%5D)">Ommochrome pigmentation of the linea and rosa seasonal forms of Precis coenia (Lepidoptera: Nymphalidae).</a> Arch. Insect. Biochem. Physiol. 36 (3), 215-222 (1997).</li>
<li>Van Den Branden, C., Decleir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Study+of+the+Chromatophore+Pigments+in+the+Skin+of+the+Cephalopod+Sepia+Officinalis%5BTitle%5D)+AND+%22L.+Biol.+Jb.+Dodonaea%22%5BJournal%5D)">A Study of the Chromatophore Pigments in the Skin of the Cephalopod Sepia Officinalis.</a> L. Biol. Jb. Dodonaea. 44 (2), 345-352 (1976).</li>
<li>Aubourg, S., Torres-Arreola, W., Trigo, M., Ezquerra-Brauer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+Jumbo+Squid+Skin+Pigment+Extract+and+its+Antioxidant+Potential+ina+Marine+Oil+System%5BTitle%5D)+AND+%22Eur.+J.+Lipid+Sci.+Technol%22%5BJournal%5D)">Characterization of Jumbo Squid Skin Pigment Extract and its Antioxidant Potential ina Marine Oil System.</a> Eur. J. Lipid Sci. Technol. 118 (0), (2016).</li>
</ol>

We have nothing to disclose.

We have demonstrated a method to extract pigments from squid chromatophore granules. By specifically targeting the granules, our goal is to determine their role in mediating adaptive coloration. This method differs from previous reports designed to characterize cephalopod pigments using bulk tissue samples14 or freeze-dried skin15.
While this protocol is effective at extracting chromatophore pigments, it is limited to small reaction scales. Dissecting one squid yields ~11...

Cephalopods such as squid, cuttlefish, and octopus have the ability to dynamically alter their appearance for camouflaging and signaling.1-6 This capability is supported in part by the selective areal expansion of pigmented organs known as chromatophores.4,7-9 Chromatophores are soft actuators that contain a network of nanostructured pigment granules confined within a cytoelastic sacculus that is anchored radially by muscle fibers.1,3 As they are actuated, chromatophores expand by 500% in presented surface area distributing the granules throughout the organ.3,7,10,11 When this action is concerted over a number of chromatophores, the overall coloration of the animal is changed. While it is known that the pigment granules contribute to this color change, their composition remains unknown. We describe a procedure to isolate and purify chromatophore pigments which may be adapted for future compositional studies.
The isolation of pigment granules involves a multistep extraction, homogenization, and purification procedure.3,12 Chromatophore containing tissue is harvested through careful extirpation from the cephalopod. A digestion and homogenization process is then used to dissociate the surrounding tissue and separate the chromatophore cells. The nanostructured granules are then isolated and purified from the remaining chromatophores using repeated sonication and centrifugation. Upon purification, pigments are extracted from the granules in a process that is adapted from the extraction of visible color from butterfly wings using acidic methanol solutions.13 Scanning electron microscopy (SEM) and spectrophotometry are used to confirm that the chromatophore pigments are successfully extracted using this process.
This method describes the isolation of chromatophore granules which is used to explore the molecular contributions to coloration in cephalopods.12 Small molecule extractions from whole animals can often be a long and tedious process. The aim here is to inform future researchers of an effective and facile protocol for the acquisition of pigments from the nanostructured granules in cephalopods.

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

Chromatophores are dissected from the D. pealeii dorsal mantle (Figure 1A, 1B). Once they are removed, chromatophores are lysed and purified using centrifugation and washing cycles to isolate out the pigmented granules (Figures 2A, 2B). Acidic methanol solutions (HCl-MeOH) are used to extract the pigment from the granules (Figure 2C), yielding a soluble pigment extract and insoluble, colorless pi...

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

The overall goal of this procedure is to describe how to extract pigments located within the nano-structured granules of freshly dissected squid, Doryteuthis pealeii chromatophores. Cephalopods are complex marine animals that undergo rapid and adaptive changes to their dermal color. and adaptive changes to their dermal color.However, there is still a lot that is unknown about how these systems work. The main advantage of this technique is that it specifically targets the nano-structured granules in the chromatophore organ. This will enable researchers to elucidate the mechanism for color change originating at the nanoscale.In addition to insight into cephalopod biology, it can inform design of synthetic systems built to mimic the dynamic range of adaptive color displayed in cephalopods using pre-packaged pigmented nano-particles. Generally, individuals new to this method struggle because collecting pure chromatophore tissue is difficult due to its proximity with the surrounding tissue. Our method is optimized to reduce these contaminants.To begin dissection, first pour approximately 250 milliliters of filtered sea water into a dissection pan. Place a specimen in the pan and using stainless steel straight fine point scissors, cut from the base of the ventral mantle along the posterior side of the animal, ending at the fin region. Remove all of the internal organs by gently lifting them with dissecting forceps and severing the connective tissue using the scissors.Discard the organs in a specimen bag. Using dissecting T pins, pin the mantle fin side up into the dissection pad. With forceps, carefully remove the opaque epidermal skin layer, keeping the chromatophore-containing skin layer intact.Discard the epidermal layer in a specimen bag. Next, remove the chromatophore layer. Cut the chromatophore tissue into small one centimeter by one centimeter sections.Place the sections into a 1.5 milliliter micro centrifuge tube, filling each tube halfway with dissected tissue. Finally, add 500 microliters of filtered sea water to the tubes. To isolate the pigment granules, first centrifuge the chromatophore tissue sections for five minutes at 14, 000 x g.Discard the top layer of seawater. Add 500 microliters of papain collagenase solution to the samples and vortex for one to two minutes on the highest setting. Next, sonicate the samples for five minutes at 40 kHz to dissociate cells from the surrounding tissue.Centrifuge for a further five minutes at 14, 000 x g and then discard the supernatant. Repeat the centrifugation a further two times, discarding the supernatant each time. Then, add the resuspension in collagenase.Use forceps to manually remove any remaining large tissue sections that did not digest before proceeding. The purity of starting granule solution influences the pigment extraction. So it is important to ensure that the starting chromatophore materials are fully digested.We find that tissue sections around one centimeter are optimal for collecting the purest population of granules. Prepare the homogenization buffer by taking a 150 milliliter screw cap tube and adding the appropriate amount of HEPES, magnesium chloride, potassium aspartate, and dithiothreitol. Add one commercial mini tablet of protease inhibitor.Using a graduated cylinder, measure 100ml of deionized water and add it to the tube containing the homogenization salts. Vortex the tube to mix until the solution becomes clear. Centrifuge the tissue samples and then discard the supernatant.Add 500 microliters of homogenization buffer to each tube and vortex to 2 minutes to thoroughly mix. Sonicate the samples for 30 minutes at 40kHz. And then centrifuge them again.After discarding the supernatant, repeat the homogenization addition, sonication, and centrifugation two to three times to ensure complete digestion of the extracellular tissue. Finally, the tube should contain a light yellow supernatant and a red colored pellet consisting of the isolated pigment granules. Discard the supernatant.First, prepare acidic methanol for extraction in a screw cap glass sample vile by adding 5ml of methanol and 25mcL of concentrated hydrochloric acid. Swirl the solution gently to mix. Set aside one sample tube, this will be the end reacted granules treatment.Remove the homogenization buffer from a second tube containing granules. Add 500mcL of the acidic methanol solution to the remaining tubes and vortex for three to four minutes. Sonicate the samples for 10 minutes at 40kHz and then centrifuge for five minutes at 14000 x g.Using a micropipette, collect the supernatant in a one-dram screw-top glass vial. This is the pigment, and it should appear dark red in color. Repeat the acidic methanol addition, sonication, and centrifugation steps four to five times collecting the supernatant until no further color is extracted.The remaining colorless pellet is the pigment extracted granules. To prepare granule samples for imaging, first centrifuge both the unreacted granule aliquot and the pigment extracted granules for five minutes at 14000 x g discard the supernatant, and then resuspend each pellet in 1ml of 95%ethanol. Using a transfer pipette, place 2-3 drops of each granule suspension onto a separate aluminum SEM stub.Place the samples in desiccator to dry overnight under vacuum. Sputter coat the stubs with gold platinum coating for 3 minutes to achieve a 15 nanometer coating. Image the samples using a scanning electron microscope.Next, use a spectrophotometer to collect the absorbance profiles for the unreacted granules, extracted granules, and extracted pigment. Take a blank measurement for the granule samples by adding 1ml of deionized water to a clean cuvette. Discard the blank into a waste container.Next, add the suspension containing the unreacted pigment granules to the cuvette. Collect the UV vis absorption spectra for the granule sample. Take a second blank measurement for the extracted pigment using 1mL of acidic methanol and a clean cuvette.Finally, take the UV vis absorbent spectrum for the pigment and then determine the maximum wavelength of each sample. Scanning electron microscopy revealed that the pigment extraction process reduces the average diameter of the granules. Prior to extraction, granules have an average diameter of 527.3nm.After the acidic methanol procedure, this size decreases significantly to around 202.6nm. A reduction thought to be due to the extraction of the pigment. Upon addition of the acidic methanol, the color associated with the unreacted granules also visibly reduces.Confirmation of this hypothesis was obtained using spectrophotometry. This graph shows a broad absorbance profile for the pre-extracted granules and a similarly broad but lower intensity profile for the post-extracted granules. In contrast, the soluble extracted pigment exhibits a lambda max at around 500nm and a distinct shoulder at around 380nm.Collectively, these data suggest the successful extraction of visible color from the granules. Once mastered, this technique can be done in approximately four hours from start to finish. While attempting this procedure it's important to remember to fully remove the tissue which contains reflective protein platelets that may interfere with the extraction and analysis.Epidermal tissue may also interfere with homogenization steps and the pigment extraction. Following this procedure, other methods like thin layer chromatography and mass spectrometry can be used to further purify the pigments and characterize their composition. These steps can help elucidate the molecular contributions to coloration in cephalopods.This method can help answer key questions for marine biologists, chemists, and engineers including:What is the composition of the pigments used during color change? And how do they contribute to the function and structure of the nano structured granules within the dermal chromatophore organs? After watching this video you should have a good understanding of how to extract pigment from freshly dissected squid, doryteuthis pealeii chromatophores.

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This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 °C.

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts' antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

Learn a method for extracting pigments from squid chromatophores using Doryteuthis pealeii. This protocol enables nanoscale analysis of color change mechanisms in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

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