This work was supported by Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (PAPIIT), with a grant number IT200819. The authors acknowledge to Tom Musselman, Rock Paper Editing, LLC, for checking the English grammar of this manuscript; and the technical assistance of Samanta Jim&#233;nez (CICESE, Ensenada), and Juan Manuel Barbosa Castillo (Instituto de Fisiolog&#237;a Celular, UNAM).&#160;We also thank to Dr Augusto C&#233;sar Lizarazo Chaparro (CEPIPSA) for obtaining sheep blood.&#160;We especially thank Dr Jos&#233; Saniger Blesa, ICAT-UNAM,&#160;&#160;for the facilities in his laboratory for the video recording.&#160;

The sea anemones were collected according to guidelines of the National Commission for Aquaculture, Fisheries, and Food of the Federal Government of Mexico (permit number PPF / DGOPTA 07332.250810.4060). Bioethics Committee of the Institute of Biotechnology, National Autonomous University of Mexico approved all the experiments with sea anemones. The sheep blood sample was purchased at the Center for Practical Teaching and Research in Animal Production and Health (CEPIPSA, National Autonomous University of Mexico).
1. Organism collection
<ol>
	<li>Collect sea anemone A. dowii. 
	NOTE: Here, the organisms were collected during low tide periods in the intertidal zone off the coast of Ensenada Baja, California, Mexico (Figure 1A,B).</li>
	<li>Transport the organisms to the laboratory in containers with seawater (Figure 1C).</li>
	<li>In the laboratory, clean the organisms with distilled water by hand to remove the big substrate particles adhering to the body.</li>
	<li>Immediately freeze the organisms at -80 &#176;C in an ultra-freezer for 72 h.</li>
	<li>Place the organisms in special glasses for freeze-drying, with lyophilization conditions of -20 &#176;C, 0.015 psi, for 48 h.</li>
	<li>Store the lyophilized organisms at -20 &#176;C until use.</li>
</ol>
2. Tissue hydration
<ol>
	<li>Reconstitute 20 g dry weight of lyophilized organisms with 60 mL (1/3 w/v) of 50 mM sodium phosphate buffer, pH 7.5, and 1 inhibitor cocktail tablet (Table of Materials).</li>
	<li>In a magnetic stirrer, continuously stir the sample at ~800 rpm, 4 &#176;C, for 12 h.</li>
</ol>
3. Toxin release
<ol>
	<li>To induce cell lysis and nematocyst discharge, freeze the sample at -20 &#176;C for 12 h, and then place the container in water at room temperature (RT) until it thaws up to 90%. Repeat this procedure three times. 
	NOTE: The sample does not thaw 100%; it is crucial to maintain the sample at ~4 &#176;C to prevent protein degradation.</li>
	<li>Observe the nematocyst discharged under a confocal microscope. Take 10 &#181;L of the sample on a slide and place a coverslip over it. Observe under a confocal microscope at 100x and 60x magnification. Here a dye was not necessary.</li>
	<li>Process the images captured in the confocal microscope in ImageJ software (version 1.53c, Wayne Rasband, National Institutes of Health, USA,&#160;https://imagej.nih.gov/ij).</li>
	<li>If the tubule inside the nematocyst uncoils and is outside, the nematocysts are discharged; then, continue with the next step. Otherwise, only conduct one more freeze-thaw cycle.</li>
	<li>To clarify the extract, centrifuge the sample at 25,400 x g for 40 min, 4 &#176;C, recover the supernatant, and centrifuge it again. Repeat this step 3-4 times.</li>
	<li>Recover the supernatant and discard the pellet. The supernatant or crude extract contains the venom components (toxins) and other cell molecules, such as lipids, carbohydrates, and nucleic acids.</li>
</ol>
4. Quantitation of total protein
<ol>
	<li>Determine the protein concentration of the crude extract using a commercial Bradford colorimetric assay15 kit (Table of Materials).</li>
	<li>Dilute the dye reagent (one part of dye with four parts of distilled water).</li>
	<li>Prepare bovine serum albumin (BSA) Fraction V (Table of Materials) solution at 1 mg/mL concentration in phosphate buffer (50 mM sodium phosphate, pH 7.5).</li>
	<li>Prepare the solutions listed in Table 1 in triplicate.</li>
	<li>Vigorously mix each solution in a shaker for 4 s.</li>
	<li>Incubate the samples for 5 min at RT, without shaking.</li>
	<li>Measure absorbance (AU) at 595 nm.</li>
	<li>Plot the averages of the sample BSA absorbances (AU vs. protein concentration), and determine the equation of the graph (Equation 1). 
	<img alt="Equation 1" src="/files/ftp_upload/63630/63630eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;Equation 1 
	where y is the absorbance, m is the slope of the line, x is the protein concentration, and b is the y-intercept. To calculate crude extract concentration, solve for the x as follows: 
	<img alt="Equation 2" src="/files/ftp_upload/63630/63630eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;Equation 2</li>
</ol>
5. Determine polypeptide venom complexity
<ol>
	<li>Assemble the glass container for vertical gel electrophoresis according to the manual.</li>
	<li>Prepare acrylamide mix (30%): 29.2 g of acrylamide, 0.8 g of bis-acrylamide, the final volume of 100 mL. Filter the solution using a 0.45 &#181;m membrane. Store the solution in a dark bottle at 4 &#176;C. Then, prepare the mixture for the resolving gel (Table 2).</li>
	<li>The SDS-PAGE gel consists of a resolving gel and a stacking gel. Prepare the gels as per the solution volumes defined in Table 2. During preparation, ensure to maintain each mix at 4 &#176;C to reduce polymerization time. 
	NOTE: The volume of each mixture varies depending on the brand of equipment used; for this protocol, the volumes correspond to the preparation of 15% acrylamide gel, 0.75 mm thick, for a vertical electrophoresis chamber (Table of Materials).</li>
	<li>After the acrylamide has polymerized, ~10 min, add the mixture of stacking gel.</li>
	<li>Prepare the mixture for the stacking gel, immediately add it to the glass plates, and place a comb to form the wells for loading the samples.</li>
	<li>Once the stacking gel has polymerized (~15 min), remove the comb and place the glass container in a chamber for vertical electrophoresis.</li>
	<li>Place 200 mL of electrode buffer (0.25 M Tris, 0.192 M glycine, 0.1% SDS) in the electrophoresis chamber.</li>
	<li>Analyze the crude extract: Mix 30 &#181;g of crude extract with 5 &#181;L of protein loading buffer (25% glycerol, 15% sodium dodecyl sulfate, 25% 2-mercaptoethanol, 0.125 mM Tris pH 7, bromophenol blue 0.1 mg/mL) to denature proteins. The final volume must be &#60;50 &#181;L.</li>
	<li>Heat at 90 &#176;C for 5 min, cool, and centrifuge for 3 s at 1,400 x g.</li>
	<li>Load the samples in the wells of the stacking gel. Load a standard molecular weight ladder.</li>
	<li>Run electrophoresis at 25 mA constant for 1 h 30 min.</li>
	<li>Remove the glass containers from the electrophoresis chamber to proceed with the staining of the gel proteins.</li>
</ol>
6. Protein staining
<ol>
	<li>Rinse the gel in 50 mL of distilled water to remove debris from the buffer electrode.</li>
	<li>Discard the water.</li>
	<li>To avoid the diffusion of the gel proteins, add the fixation solution (60 mL of ethanol, 20 mL of acetic acid, and 20 mL of distilled water, final volume 100 mL). Incubate at RT for 40 min with shaking at 80 rpm. Decant the solution.</li>
	<li>Add the protein crosslinking solution (15 mL of ethanol, 0.25 mL of glutaraldehyde, 50 mL of distilled water, final volume 65.25 mL), and incubate for 30 min at RT with shaking at 80 rpm. Remove the solution.</li>
	<li>Wash the gel with 100 mL of distilled water for 5 min, and remove the water. Repeat this procedure four times.</li>
	<li>Carry out protein staining with 50 mL of Coomassie brilliant blue R-250 filtered solution (40% methanol, 10% acetic acid, 50% distilled water, 0.1% dye, final volume 100 mL), stirring at 60 rpm in a laboratory rotary oscillator at RT for 15 min.</li>
	<li>Recover the solution, which can be used to stain other gels.</li>
	<li>To eliminate the excess dye, add a destaining solution (40 mL of methanol, 10 mL of acetic acid, 50 mL of distilled water). Keep stirring (80 rpm) at RT until the bands (stained proteins) visualize.</li>
	<li>Keep the gel in 50 mL of distilled water and scan the image in a Gel documentation system.</li>
</ol>
7. Prepare red blood cell solution
<ol>
	<li>Extract 1 mL of blood from the jugular vein of a sheep.</li>
	<li>Remove the needle from the syringe and immediately drain blood into a tube with 50 mL of Alsever solution (0.002 M citric acid, 0.07 M NaCl, 0.1 M dextrose, and 0.027 M sodium citrate as an anticoagulant, pH 7.4).</li>
	<li>Slowly invert the solution three times.</li>
	<li>Keep at 4 &#176;C for transfer to the laboratory.</li>
	<li>Centrifuge at 804 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet in 30 mL of Alsever solution. Add the solution by sliding it along the tube&#39;s inner walls and, using a Pasteur plastic pipette, slowly resuspend the cells. Centrifuge again at 804 x g for 5 min, 4 &#176;C. Repeat this procedure until the supernatant is clarified.</li>
	<li>Add 15 mL of Alsever solution; resuspend the pellet slowly with a Pasteur plastic pipette.</li>
	<li>Maintain the final volume of Alsever solution to achieve a concentration of 2 x 106 cells/mL, approximately. Then, use a Neubauer chamber or hemocytometer to count the cells. 
	NOTE: Count the erythrocytes in the hemacytometer slide (Improved Neubauer). The slide is a 30 mm x 70 mm x 4 mm slide. The center has two silver footplates (Figure 2A) (one top and one bottom grid), each 3 mm x 3 mm, and two channels on the sides of the grid. Counting cells is in the corner and center squares (red squares in Figure 2B). The optimal concentration of cells for counting should be in the order of 106 cells. The central box has a surface area of 0.1 cm2, which is equivalent to 0.0001 mL.</li>
	<li>Place a coverslip over the slide.</li>
	<li>Place 10 &#181;L of the sample between the slide and the coverslip and wait for the sample to disburse.</li>
	<li>In an optical microscope (magnification 60x), perform the count in the first square box. Count only the cells in the center grid and those that touch the upper or the left margin of the box.</li>
	<li>Determine cell concentration using the following equation: 
	<img alt="Equation 3" src="/files/ftp_upload/63630/63630eq03v4.jpg" style="margin: auto;" /> Equation 3 
	If the sample is very concentrated and dilution is required, then use the following equation: 
	<img alt="Equation 4" src="/files/ftp_upload/63630/63630eq04v3.jpg" style="margin: auto;" /> Equation 4 
	NOTE: When taking the erythrocyte solution for the hemolysis test, slowly homogenize the solution with a plastic Pasteur pipette.</li>
	<li>Perform the steps in triplicate to reduce error.</li>
</ol>
8. Hemolysis assay
<ol>
	<li>Prepare the solution mixtures (in triplicates) indicated in Table 3 in conical 96-well plates. 
	NOTE: Mix the erythrocytes slowly to maintain a homogeneous suspension.</li>
	<li>Incubate for 1 h at 37 &#176;C, without shaking.</li>
	<li>Centrifuge the plate at 804 x g for 5 min at 4 &#176;C.</li>
	<li>Recover the supernatant in another 96-well plate with a flat bottom.</li>
	<li>If the crude extract contains cytolysins, erythrocytes will lyse and release hemoglobin. Read the absorbance at 415 nm.</li>
	<li>Calculate hemolysis percentage, using the following equation: 
	%&#160;Hemolysis = ((Acrude extract-AAlsever buffer) / (AH2O-AAlsever)) x 100 
	NOTE: Acrude extract, AAlsever buffer, and AH2O correspond to the absorbance of erythrocytes with the crude extract, absorbance of Alsever solution, and absorbance of distilled water, respectively.</li>
	<li>Perform a hemolysis test before and after each freezing and thawing cycle with 50 &#181;g of total protein from the crude extract.</li>
	<li>Determine the number of freezing and thawing cycles required to reach 100% hemolytic activity.</li>
	<li>Calculate the amount of extract that produces hemolysis in 50% of the erythrocytes (HU50); follow the same protocol described in Table 3, increase the amount of sea anemone crude extract, and design the plot with sigmoidal adjustments using appropriate software (e.g., Origin, Table of Materials).</li>
</ol>
9. Phospholipase assay
<ol>
	<li>Wash one chicken egg with 1% SDS in distilled water.</li>
	<li>Separate the egg yolk from the egg white under sterile conditions.</li>
	<li>Prepare 50 mL of 0.86% NaCl solution, and filter through a 0.22 &#181;m filter. Then, prepare Solution A: Add 12 mL of egg yolk and 36 mL of 0.86% NaCl solution.</li>
	<li>Prepare Solution B: Mix 300 mg of agarose in 50 mL of buffer (50 mM Tris, pH 7.5), filter through a 0.22 &#181;m filter. Heat the solution in a microwave until boiling. Cool down in warm water to reach 43-45&#176;C.</li>
	<li>Prepare Solution C: Prepare 10 mM CaCl2 and filter it with a 0.22 &#181;m filter.</li>
	<li>Prepare Solution D: 10 mg of rhodamine 6G in 1 mL of distilled water.</li>
	<li>Under sterile conditions (laminar flow hood), add 500 &#181;L of solutions A and C to solution B and 100 &#181;L of solution D, mix, and pour 25 mL into each Petri dish (90 x 15 mm).</li>
	<li>Wait for the solution to solidify under sterile conditions (30 min).</li>
	<li>Make wells (~2-3 mm diameter) with a thin tube.</li>
	<li>Add a total of 20 &#181;L of phosphate buffer (negative control) in one well and 20 &#181;L of a determined phospholipase (positive control) in another well.</li>
	<li>Place different amounts of the crude extract protein, 5, 15, 25, 35, 45 &#181;g, in the remaining wells, each in a final volume of 20 &#181;L.</li>
	<li>Wait for the agar to adsorb all the samples (30 min).</li>
	<li>Incubate at 37 &#176;C for 20 h.</li>
	<li>If a halo forms around the well, this indicates phospholipase activity. Measure the halo formed with a vernier caliper. 
	NOTE: Perform the experiment in triplicate (steps from 9.1-9.14).</li>
</ol>

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The authors declare that they have no competing interest in relation to the publication of this paper.

The high demand for new compounds with applications in different fields of science and industry has led to the study of venom. Venom represents a rich source of molecules that serves as a template for generating new molecular tools. However, the complexity of these venoms requires the implementation and combination of various methods to obtain and study them.
Here, we show a method for obtaining and analyzing the venom of the sea anemone Anthopleura dowii, Verrill 1869, which can be u...

Marine animals are a rich source of biologically active compounds. In recent decades, the composition of sea anemone venom has attracted scientific attention since it comprises a diversity of polypeptides with hemolytic, cytotoxic, enzymatic (phospholipase, protease, chitinase), and neurotoxic activity and inhibitory effects on proteolytic activity1. In addition, these polypeptides are potential sources for the development of molecular tools in biotechnological and therapeutical use2,3.
There are few reports about sea anemone venom and its molecular components due to the complexity of obtaining the venom, even isolation, and characterization of toxins. The extraction methods used in the reports involved lysis and emptying the contents of cells that are related and unrelated to the venom production1.
A particular characteristic in all cnidarians is the absence of a system for production and release of the venom centralized in a single anatomical region. Instead, the nematocysts are structures that keep the venom4,5. Other types of cells, called epidermal gland cells, also secrete toxins and are also distributed throughout the body of sea anemones6.
The first and most crucial challenge in obtaining the venom is the generation of an extract with sufficient manipulation in subsequent processes, without the inactivation or degradation of labile proteins. Next, the cells must be lysed, and the components-in this case, polypeptides must be efficiently and quickly extracted, avoiding proteolysis and hydrolysis while eliminating other cellular components7.
Different methods are used to obtain the crude extract of a sea anemone; some involve sacrificing the organism while others allow it to be kept alive. Methods that imply the use of the organism&#180;s whole body allow for the release of most toxins from the venom8, compared to methods that keep organisms alive, which extract only some components of the venom9. The preparation of an extract requires evaluating the presence and potency of a substance of interest through a specific bioassay, which includes strategies to observe the pharmacological effects by in vivo or in vitro methods10.
Sea anemone venom contains cytolytic polypeptides, pore-forming toxins (PFTs)11, and phospholipases12; these molecules are models in the study of protein-lipid interaction, molecular tools in cancer therapy, and biosensors based on nanopore3. The classification of sea anemone PFTs is carried out according to their size or molecular weight, from 5 kDa to 80 kDa. The 20 kDa PFT, the most studied and known as actinoporins11, is of particular interest for its biomedical potential in the development of molecular tools for possible applications as anticancer, antimicrobial, and nanopore-based biosensors. Another cytolysin, including phospholipases, specifically phospholipase A2 (PLA2)13, releases a fatty acid due and hydrolyzes phospholipids, destabilizing the cell membrane. Due to this mechanism of action, PLA2 promises to be an essential model for the study and applications in inflammatory diseases. It could serve as a model for studies of lipid behavior in the cell membrane14.
Here, we describe an efficient protocol for obtaining the crude extract from sea anemone Anthopleura dowii Verrill, 1869, and detecting hemolysins and phospholipases. Both are relevant toxins that could be used as a template to design new molecular tools.

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			<td>15 mL conical centrifuge tube</td>
			<td>Corning</td>
			<td>430766</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Bromophenol blue</td>
			<td>Sigma</td>
			<td>B75808</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoetanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>J.T. Baker</td>
			<td>9515-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Promega</td>
			<td>V3115</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisacrylamide</td>
			<td>Promega</td>
			<td>V3143</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Fraction V</td>
			<td>Sigma</td>
			<td>A3059-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Protein Assays</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates 96 well, V-bottom</td>
			<td>Corning</td>
			<td>3894</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Corning</td>
			<td>CLS430829</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc MP system</td>
			<td>Bio-Rad</td>
			<td>1708280</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear flat.bottom 96-Well Plates</td>
			<td>Thermo Scientific</td>
			<td>3855</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue G-250</td>
			<td>Bio-Rad</td>
			<td>#1610406</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R-250</td>
			<td>Bio-Rad</td>
			<td>1610400</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>J.T. Baker</td>
			<td>1916-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ductless Enclosure</td>
			<td>Labconco</td>
			<td>Vertical</td>
			<td>https://imagej.nih.gov/ij ImageJ 1.53c</td>
		</tr>
		<tr>
			<td>Gel Doc EZ</td>
			<td>Bio Rad.</td>
			<td></td>
			<td>Gel Documentation System</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Marienfeld</td>
			<td>650030</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ (Software)</td>
			<td>NIH, USA</td>
			<td>Version 1.53c</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator 211</td>
			<td>Labnet</td>
			<td>I5211 DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T. Baker</td>
			<td>9049-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN tetra cell</td>
			<td>Bio-Rad</td>
			<td>1658000EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T. Baker</td>
			<td>3824-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>J.T. Baker</td>
			<td>3624-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>J.T. Baker</td>
			<td>3818-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Origin software</td>
			<td></td>
			<td>version 9</td>
			<td>To design the plot with sigmoidal adjustments</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman kit</td>
			<td>Gilson</td>
			<td>F167380</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast mini gel</td>
			<td>BioRad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained Protein Ladder</td>
			<td>Thermo Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Assay Dye Reagent Concentrate</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma-Aldrich</td>
			<td>252433</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L4509</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>JT Baker</td>
			<td>3646-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>THERMO SCIENTIFIC</td>
			<td>G10S UV-VIS</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Volt Power Supply</td>
			<td>Hoefer</td>
			<td>PS300B</td>
			<td></td>
		</tr>
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</table>

identification of hemolytic and phospholipase activity in crude extracts from sea anemones by straightforward bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

The representative results of the protocol used to obtain the crude extract of sea anemone showed that combining two techniques (agitation and cycles of freezing and thawing) produced an efficient discharge of nematocysts, and the total amount of protein was 500 mg (8 mg/mL) (Figure 3).
The crude extract&#39;s protein complexity could be observed from 10 kDa and greater than 250 kDa through SDS-PAGE electrophoresis. In addition, cytolysins were detected in the mol...

Watch this Scientific Journal Video about Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays at JoVE.com

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

identification-hemolytic-phospholipase-activity-crude-extracts-from

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Biochemistry

2.6K Views.  Universidad Nacional Autónoma de México. Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Video: Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...