Here we describe how to prepare the biological material for proteomic analysis, including the appropriate kits, type of columns and technologies needed for obtaining and processing crude extracts. Bioassays requiring highly specialized facilities or authorizations will not be contemplated. Sea anemones and jellyfish procedures were reviewed previously in 2012  and 2004 , respectively.
Here we reviewed relevant information on several jellyfish species including Aurelia aurita [8,9,10,11], Carybdea alata , Carybdea rastoni , Chironex fleckeri [14,15,16,17,18,19], Chiropsalmus quadrigatus , Chiropsalmus sp. , Chiropsella bronzie , Cyanea capillata [21,22], Cyanea nozakii [11,23,24], Nemopilema nomurai [11,25,26], Pelagia noctiluca [27,28,29,30,31,32,33], Rhopilema esculentum [11,34,35], Rhopilema nomadica , Stomolophus meleagris [37,38,39,40,41] and deep-sea jellyfish . Apart from the true jellyfish, there are also Hydrozoa members that have different evolutionary histories  and are colonial free-swimming organisms resembling jellyfish. Olindias sambaquiensis [44,45] and Physalia physalis  are two of such examples. When considering jellyfish sample collection, the most important step is to quickly preserve proteins from any kind of degradation. For that purpose, all the procedures should be kept in ice to avoid loss of efficacy , or they should proceed as explained by the authors. Jellyfish can be transported live to the laboratory in plastic buckets with seawater  or processed in the field. Tentacles can be immediately processed and stored in seawater at 4 °C  prior to nematocyst extraction. Alternatively, the tissues can be immediately stored at −20 °C . The transport of jellyfish to the laboratory facility should consider the use of ice  or dry ice [12,13,20]. Dry ice  or lyophilized material  is also suitable for shipping material. Another option is to snap-freeze tentacles in liquid nitrogen and store them at −80 °C [12,13,37,38] until use.
2.1. Nematocyst Extraction
The nematocyst extraction can be accomplished using several protocols. One of the most commonly used methods is that proposed by Bloom et al. , which requires refrigerating the tentacles in two volumes of seawater for one to four days. Once a day the containers should be vigorously shaken and an aliquot filtered through a fine kitchen sieve. The decision on whether or not to continue digestion of individual samples is based on microscopic examination of the tentacles and the filtered sediment. If digestion is to be continued, the original fluid is decanted, replaced with fresh seawater, and the filtered material washed with seawater, allowing three hour-long settling periods under refrigeration. The final sediments are then lyophilized and stored at −80 °C until use.
Several works [17,19,26,47] used this well-established method (or with slight adaptations). Feng et al.  has frozen the tentacles placed in 5× vol. of fresh seawater at 4 °C and set the autolysis for four days. The resulting suspension was filtered through a fine sieve (54 mm). The sediments (where most of the nematocysts are contained) were then collected and washed several times with 0.9% NaCl solution. Xiao et al.  set the mass:volume ratio to 1:1. The mixture was stirred for 30 min twice daily and the autolyzed mixture was centrifuged thrice. Nagai et al.  isolated the nematocysts using a modification of the Burnett method . After four days, the sample was filtered through a nylon stocking and the nematocysts in the filtrate were allowed to settle in a 50 mL conical centrifuge tube. More recently, the method has been simplified and performed faster [24,37] with an overnight autolysis at 4 °C, and afterwards the tentacles are stirred for 10 min and filtered through a plankton net (approximately 280 µm) to remove most of the tentacle debris. Brinkman et al.  purified the nematocysts from tentacle debris by centrifugation in a discontinuous Percoll gradient diluted with 35 g/L NaCl.
In other works [27,28,29,30,32], the nematocysts were isolated as described by Salleo et al. . Briefly, the oral arms were excised and submerged in distilled water for 2 h at 4 °C. After a complete detachment of the epidermis, the tissue was removed from the suspension, containing both epidermis and undischarged nematocysts (organoids) deriving from the osmotic rupture/lysis of nematocytes. The nematocysts that were still attached to the epidermal tissue were separated by stirring. The nematocyst suspension was repeatedly washed in distilled water and filtered through plankton nets to remove most of the tissue debris, and then centrifuged. In a similar way, Lee et al.  and Kang et al. [25,51] gently swirled the tentacles with distilled water, and then stood still for 1–2 h to remove debris and seawater. After decanting the supernatant, tentacle pellets settled down at the bottom were resuspended in about a 2× vol. of distilled water and shaken vigorously for 3 min. However, in this procedure the detached nematocysts were separated by filtering the tentacle preparation through four layers of medical gauze.
In the study of the tentacle venom fraction from the jellyfish Aurelia aurita , cold deionized water and a protease inhibitor cocktail was used for nematocyst extraction. Previously, Gusmani et al.  used the scapular and mouth filaments suspended in cold sterilized distilled water containing 1 M glycerol for 2 h at 5 °C, and then stirred it. The resulting suspension was filtered through a plankton net and the mesogleal component discarded. After centrifugation the supernatant was discarded and the pellet was resuspended in the same medium. The procedure was repeated twice and the final suspension containing undischarged nematocysts was deep-frozen for further use. In brief, autolysis can be performed in fresh seawater, artificial seawater, filtered seawater or reverse-osmosis purified water to different concentrations of saline solution [10,14,22].
2.1.2. Alternative to Autolysis
As an alternative to autolysis with different types of water, Weston et al.  isolated the nematocysts using a modification of the Weber et al.  method. The tentacles were gently homogenized in a pestle and mortar in cold SuFi (sucrose + Ficoll-Paque Plus) solution. The material was kept at 4 °C for 30 min and then passed through a 2 mm diameter sieve. The sample was centrifuged and the supernatant containing debris and cell fragments was removed. The pellet containing intact nematocysts was carefully suspended and washed three times in cold SuFi solution. The final material was submitted, after microscopic inspection, for lyophilization. In a different approach, Ovchinnikova et al.  excised pieces of Aurelia aurita and placed them for extraction into 5% of acetic acid. The obtained extract was consecutively passed under pressure, three times, through a stirred ultrafiltration cell and dried in vacuum. On the other hand, Radwan et al.  attempted to dissociate the nematocysts from both tentacle tissue and dinofagellates by renograffin density gradient centrifugation according to Calon et al. .
The simpler the method, the better, as long as it performs well. Indeed, simpler methods are faster and less susceptible to errors. Autolysis overnight at 4 °C, tentacles stirred for 10 min and filtered through a plankton net are suitable to obtain a nematocyst solution.
2.2. Venom Extraction
For venom extraction several protocols can be used. Venom can be obtained by sonication of the nematocyst suspension in cold extraction buffer [24,27,28,29,30,37,38,39] followed by centrifugation. Bloom et al.  proposed a widely used protocol. Freeze-dried samples are resuspended in 1:6 with cold deionized water and sonicated for three 20 s periods. Sonications are intercalated with cooling periods of at least 1 min on ice. The degree of nematocyst rupture is examined microscopically. The suspension is clarified by centrifugation at 20,000× g for 1 h at 4 °C, being the supernatant composed of the nematocyst proteins.
Nematocyst sonication can be performed in the presence of different buffers. Weston et al. , disrupted in a sonic bath, freeze-dried nematocysts in TEAB (Triethylammonium bicarbonate buffer) whereas Gusmani et al.  sonicated the nematocysts in acetate buffer containing NaCl and the protease inhibitors benzamidine and iodoacetic acid. Nagai et al. [12,13] sonicated nematocysts and frozen tentacles in a phosphate buffer solution.
2.2.2. Nematocyst Mechanical Disruption
Extraction of venom according to Carrette and Seymour  using glass beads in an ice-cold (4 °C) solution has been adopted by several authors [11,14,25,26]. The samples incubated with PBS solution were shaken in a mini-bead mill with intervals five times with intermittent cooling on ice. The venom extract was then transferred to a new Eppendorf® tube and centrifuged. Both the supernatant and the venom were used. Winter et al. [17,18] applied this method, but replaced the PBS solution with distilled water. In a different approach, Brinkman et al. [7,15] resuspended the nematocysts in ice-cold buffer (MOPS, NaCl and protease inhibitors) and ruptured them using 0.5 mm glass beads. Nematocyst disruption was monitored microscopically and >90% rupture of nematocysts was achieved with four to six cycles of homogenization. Crude nematocyst extracts were clarified by centrifugation. In the case of Feng et al.  lyophilized nematocysts were placed into screw-top vials with Tris-HCl buffer and glass beads. Samples were shaken four times in a mini–bead beater with intervals on ice. The venom samples were then separated from the glass beads with a pipette and centrifuged.
Pestle and mortar can be used to homogenize tentacles for venom studies . Kawabata et al.  crushed them with PBS. The samples were then centrifuged and the supernatant was filtered through a cellulose acetate membrane filter to obtain the crude extract. A blender can also be used with ice-cold water . In this procedure the crude lysate was clarified through a layer of spun glass and subsequently centrifuged. Using 0.1% trifluoroacetic acid as an incubation solution, Junior et al.  were able to obtain venom from Olindias sambaquiensis tentacles. After freeze-thaw cycles and centrifugation, the supernatant was recovered and filtered through a 0.45 μm filter, followed by a second ultrafiltration using a 0.22 μm filter.
When the whole body of the jellyfish is used to obtain the venom content, mincing can be used to obtaining the venom. Another approach was used by Brinkman et al. . For venom collection, Percoll-cleaned nematocysts were washed with Tris-HCl and resuspended in SDS-sample buffer containing DTT (Dithiothreitol) until nematocyst discharge.
The accumulated knowledge revealed the use of glass beads as the most effective approach. Compared to sonication, both techniques require special equipment, but when using glass beads the risk of sample contamination is minor and the time to rupture the cells is less, therefore causing reduced protein degradation when rupturing the nematocysts. The use of a blender or a mortar and pestle also increases the risk of protein degradation and contamination compared to glass beads.
Concentration of Venom Proteins
The concentration of total protein in venom extracts can be determined with the Bradford method [19,54], or by measuring absorbance at 280 nm and using bovine serum albumin as a reference standard  or by a Pierce BCA assay kit [9,12,13,20,26,46,55]. Alternatively, Nanodrop™ also estimates total protein from extracts . Winter et al.  have quantified the amount of protein obtained after filtering the supernatant through a micro 0.2 µm syringe filter, read at 562 nm in a Fusion α microplate reader. Other authors quantified the proteins based on the techniques of Waddell  or Lowry et al. . For the identification and quantification of the different venom components, immunodetection (ELISA and Western blot) or proteomics methodologies combining sample fractionation (liquid chromatography and/or gel electrophoresis) and mass spectrometry could be used. These methodologies are explained in more detail below.
2.3. Toxin Purification, Detection and Identification
For toxin purification and identification, several methods can be applied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE), gel filtration, liquid chromatography-mass spectrometry (LC-MS), reverse-phase high performance liquid chromatography (RP-HPLC), DEAE-Sepharose Fast Flow anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) are commonly used.
Gel electrophoresis allows the discrimination of proteins according to size or purification of a protein extract by picking proteins out of the gel. Briefly, samples are mixed with 4× loading buffer (Tris-HCl, SDS, glycerol, bromophenol blue, β-mercaptoethanol). The samples are reduced at 100 °C and then cooled. The gels of polyacrylamide contained acrylamide/bis solution, Tris-HCl, SDS, TMED and ammonium persulphate. Broad range molecular weight markers are used for quantification. The electrophoretic buffer contains Tris base, glycine and SDS. The protein bands are visualized with Coomassie R-250 staining [15,22], silver nitrate [15,38,39] or both to elucidate any bands that could be missed with just one stain . The gels are then destained in KCl. This SDS-PAGE procedure description follows Laemmli [22,58]. Other authors [23,24] make a SDS-PAGE with slight modifications, namely a sample dilution (v/v = 1:1) with 2× loading buffer. Prior to 2DE, the venom could be treated with cold acetone for 2 h. The samples are centrifuged and the pellets dried in the lyophilizer. The dried pellets are dissolved in the sample buffer (containing urea, thiourea, CHAPS, DTT and IPG buffer) and afterwards in rehydration buffer (containing the previously described reagents plus bromophenol blue) and then applied to an IPG (Immobilized pH Gradient) strip. After isoelectric focusing (IEF), the IPG strips are first equilibrated with DTT and then with iodoacetamide. Thereafter, the IPG strips are placed onto 12% SDS-PAGE gel and sealed with agarose, before beginning the run .
According to several authors, jellyfish venoms assessed by SDS-PAGE [8,9,10,11,12,13,14,15,18,20,24,25,27,33,39,46,47,56] can be analyzed using diverse percentages of polyacrylamide. In a staking gel, it can range from 4% to 5% [10,23,24], and in a separating gel from 10% to 20% [10,15,16,23,24,44]. Gusmani et al.  made a SDS-PAGE on a continuous gel gradient from 6% to 15% polyacrylamide and Ovchinnikova et al.  applied a preparative continuous acid-urea-PAGE. Tricine gels are also used to detect smaller-sized proteins compared to glycine gels . A Native PAGE analysis [41,59] can also be made. In this case, the SDS is abolished from the resolving gel and electrophoretic buffer, while the loading buffer lacks β-mercaptoethanol and SDS.
2.3.2. Gel Extraction
Proteins can be obtained through in-gel digestion. Briefly, after electrophoresis each band (SDS-PAGE) or dot (2DE) of interest is excised. In-gel reduction, alkylation, and proteolytic digestion with trypsin are performed for each band/dot gel piece . After digestion, the supernatants are recovered with formic acid and acetonitrile (ACN). The extracts are dried in a vacuum centrifuge [16,25]. Other authors [37,38] digested the venom prior to any separation method. The lyophilized venom is dissolved in guanidine hydrochloride with DTT in Tris-HCl and then reacted at 37 °C. Subsequently, iodoacetic acid is added and reacted at room temperature in the dark. NH4HCO3 is added and then centrifuged with an ultrafiltration device (Molecular Weight Cut Off—3 kDa). The sample is then digested with trypsin for 20 h at 37 °C. Finally, the digested proteins are lyophilized and stored at −80 °C until use. Brinkman et al.  used OFFGEL electrophoresis (OGE). Tryptic digested fragments were diluted in peptide-focusing buffer, without the addition of ampholytes. The samples were focused and peptide fractions were harvest and lyophilized.
2.3.3. Gel Filtration and Columns
Gel filtration is a size exclusion technique. It includes media such as Sephacryl and Superose (e.g., 12 HR (high resolution) ) for wide ranges of fractionation, Superdex for high resolution, and Sephadex (e.g., G-75 ; G-50M ) for small molecules in organic solvents.
Sephadex G-50 M equilibrated with acetic acid was used by Sánches-Rodriguez and Lucio-Martinez . Thereafter, the active fraction was passed through a QAE Sephadex A-25 column and subsequently through a Fractogel EMD SO3− column equilibrated with ammonium acetate. Afterwards, the sample was concentrated under vacuum and desalted in a Sephadex G-25 column.
Gel filtration could be a following procedure after ion-exchange chromatography, which allows the separation of proteins based on their affinity to the ion exchanger. Indeed, Feng et al.  used a Sephadex G-100 column pre-equilibrated with Tris-HCl buffer, Li et al.  used a Superdex 75 column pre-equilibrated with a size-exclusion standard mixture (SERVA) or a Superdex 200 column  and Chaoussis et al.  used a Superdex 200 10/300 GL Column in Fast Protein Liquid Chromatography (FPLC). This last work  wanted to select proteins in a range of 600 kDa–10 kDa, and, therefore, prior to sample running, a standard curve was generated with manufacturer proteins to allow determination of the proteins’ molecular weights. Subsequently, crude venom was dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS) and run through the column for eluting fractions in a 96-well plate. Winter et al.  used a Superdex S-200 column with PBS solution as the mobile phase. The eluant was monitored at 280 nm. Using similar columns, Radwan et al.  performed venom elution in a Sephadex G-200 and afterwards in a Sephacryl S-200 HP prepacked column. Proteins were eluted in phosphate buffer. In a different approach, Li et al.  filtered the solution of interest with a 0.45 µm filter membrane and loaded it onto a Superdex 75 column preequilibrated with NaCl in PBS. The purified protein was subjected to a TSK gel G3000PWxL column. Diaz-Garcia et al.  purified proteins by resuspending the lyophilized crude extract in ammonium acetate following filtration through a 0.22 µm membrane. Aliquots were subjected to gel filtration on a Superdex 75 column. The column was previously equilibrated and eluted with the sample buffer. The low molecular weight fractions of interest were pooled and loaded onto a Phenomenex ODS-3 reverse phase column. Components were eluted using a linear gradient of ACN:TFA (acetonitrile:trifluoroacetic acid). All columns mentioned were connected to a HPLC system.
2.3.4. Fast Flow Anion-Exchange Chromatography
DEAE-Sepharose Fast Flow anion-exchange chromatography is a technique frequently employed for toxin purification. Feng et al.  pre-equilibrated the column with Tris-HCl buffer, and the nematocyst extract was ultrafiltered using a 10 kDa membrane. The column was stepwise-eluted using a NaCl gradient ranging from 0.1 to 0.6 M in the equilibrating buffer. Fractions showing lethal activities in the bioassay were collected, pooled, concentrated by ultrafiltration and proceeded to gel filtration. For isolation of the hemolytic proteins from the nematocyst venom of the jellyfish Stomolophus meleagris , the hemolytic fractions of the dialyzed venom sample were loaded onto a 60 mL DEAE Sepharose Fast Flow column. Afterwards, the eluates were pooled and concentrated by a 3 kDa ultrafiltration device and went for a gel filtration. In another study, Li et al.  applied the crude venom to a 16/20 ion-exchange chromatography column packed with DEAE-Sepharose Fast Flow, which was pre-equilibrated and connected to a UV-ribonucleoprotein detector. After removing unbound proteins with EBS, the column was then eluted using stepwise increases in NaCl in Tris-HCl. The eluted fractions were monitored by UV detection and were then collected. Fractions of the same peak were pooled and concentrated using a 3 kDa Ultra centrifugal filter device and subjected to a hemolytic activity test. Thereafter, the toxic fractions were purificated by gel filtration.
HPLC is a technique that purifies more or less complex protein samples. It can be used as one of the first techniques for purifying proteins or it can be used after more coarse methods, such as gel filtration and toxicity assays, as explained in the following examples. Ponce et al.  performed a chromatography fractionation of the venom in a Vydac C18 analytical RP-HPLC column. Maisano et al.  inserted its extracts in a BioSuite 250, 10 µm SEC, 7.5 × 300 mm column. Nagai et al. [12,13,20,55] applied venom extracts first to an ion-exchange HPLC, TSK-GEL CM-650S column and then to a TSK-GEL CM-5PW column. Throughout the purification process, each fraction was checked for its hemolytic activity and pooled. The concentrated sample was applied to a gel-permeation HPLC, Superdex 75 column. Sánchez-Rodríguez  determined the purity of the protein fraction by HPLC on a Varian ProStar 410 Autosampler using a Nucleosil C18 reversed phase column. Ovchinnikova et al.  purified by RP-HPLC on a Macrosphere 300 C-18 column, with active fractions in microbes. Nagai et al.  incubated the toxins first with lysylendopeptidase and then applied the samples onto a reversed-phase HPLC column: Bakerbond wide-pore ODS 5 µm with MeCN:TFA as solvent system.
2.3.6. Mass Spectrometry
Besides HPLC, nano liquid chromatography (nanoLC-MS/MS) has been also successfully applied in other studies. LC-MS/MS is one of the most powerful techniques in the field of proteomics, allowing high-throughput identification of proteins out of complex protein mixtures. Li et al. [37,38], for the identification of venom extracted from the jellyfish Stomolophus meleagris, desalted the digested proteins on reversed phase columns (Zorbax 300 SB C18), which were then separated with an analytical RP (Reverse phase) column using the Ettan MDLC system. The process of separation used a Finnigan LTQ linear ion trap MS equipped with an electrospray interface connected to the LC setup for eluted peptide detection. Brinkman et al. , for protein identification, used a Dionex Ultimate 3000 HPLC with an Agilent Zorbax 300SB-C18 column. Eluates from the RP-HPLC column were directly introduced into the NanoSpray II ionization source of a QSTAR Elite Hybrid MS/MS System operated in positive ion electrospray mode. Weston et al.  reconstituted the protein extract in ammonium bicarbonate prior to LC-MS/MS analysis and samples were analyzed on a Thermo Scientific Orbitrap Velos Pro mass spectrometer coupled to an EASY-nLC II (Proxeon) nano LC system. After mass spectrometric analysis, liquid chromatographic separation was performed. Samples were trapped on an Easy-column packed with ReproSil-Pur C18 (3 µm). In these procedures the separations were performed with a gradient of formic acid: ACN.
MALDI-ToF allows the identification of peptides and is commonly applied to jellyfish venom analysis . Li et al.  made a Peptide Mass Fingerprinting (PMF) analysis using a MALDI-ToF-MS, where the N-terminal amino acid sequence was determined by the method of Edman degradation. Diaz-Garcia et al.  used this same method and an automatic gas-phase protein sequencer. Another example of the application of this technique is the peptide molecular mass determination described by Ovchinnikova et al. , where peptide microsequencing was made in the Procise cLC 491 Protein Sequencing System. Likewise, Brinkman et al.  determined the internal amino acid sequences for two jellyfish toxins. Nagai et al.  used this technique for peptide mapping of toxins, where separated peptide fragments were fractionated and their amino acid sequences analyzed by a PSQ-1 protein sequencer.
For protein purification, detection and identification, gel electrophoresis is a technique transverse to all types of works. It does not require expensive equipment, and is fast and most informative. For the separation of toxin fragments, gel filtration is one of the most employed and easy-to-use techniques. Other methodologies referred to here require more specialized skills and their selection should be based on study purpose and easy access to that equipment.
2.3.7. Glycoproteins, Phosphoproteins and Antioxidant Protein Detection
Brinkman et al.  were able to detect glycoproteins and phosphoproteins using a GlycoProfile III fluorescent glycoprotein detection kit and a Pro-Q Diamond phosphoprotein gel stain, respectively. For glycoprotein analysis, a duplicate gel was processed, omitting the oxidation step to detect any non-specific fluorescent staining. For phosphoprotein analysis, the ProteoProfile PTM marker containing phosphorylated ovalbumin and β-casein was included as a positive control. Fluorescently stained glycoproteins and phosphoproteins were visualized using a ChemiSmart 3000 image acquisition system.
For the purification of an antioxidant protein, Li et al.  used an ammonium sulfate precipitation procedure. For that purpose, the nematocysts were sonicated in cold extraction buffer with sodium EDTA, PMSF, pepstatin A, leupeptin and aprotinin in PBS. Ammonium sulfate was added into aliquots of seven groups representing saturation from 20% to 80%. The precipitation was dissolved and dialyzed for further analysis .
2.3.8. Western Blot Analysis
In a Chironex fleckeri study  to detect the proteins of interest (CfTX-1 and CfTX-2), as well as in other works [16,25], a Western blot analysis was performed using polyclonal antibodies purchased or raised in rabbits. The nematocyst extract proteins separated by SDS-PAGE were transferred to Immobilon-P® membranes. Membranes were blocked in non-fat milk powder in TBST and incubated overnight with either purchased antibodies or rabbit antiserum diluted in blocking solution. Membranes were washed in TBST, then incubated with secondary antibodies, conjugated to alkaline phosphatase and diluted in TBST. Following membrane washing, antigenic proteins were visualized using NBT/BCIP . The antibody-containing serum was analyzed by ELISA assay . Nagai et al.  also completed their study with a Western immunoblotting assay. The blots were saturated with a 5% skim milk solution in phosphate buffer containing Tween 20, and reacted with the polyclonal antiserum against the toxin-fragmented peptide. The bands were revealed using a peroxidase-conjugated anti-rabbit serum and the Western blotting detection reagent ECL Plus.
2.4. Toxicity Assays and Others
2.4.1. Venom Proteolytic Activity
Gusmani et al.  and Lee et al.  assessed the proteolytic activity of jellyfish venom with gelatin, casein, and fibrin as substrates that were dissolved in sodium phosphate buffer and used in 15% polyacrylamide zymography gels. Venom extracts were prepared in non-reducing sample buffer, and then run on gels at 4 °C.
After electrophoresis, SDS was removed by washing the gel twice in Triton X-100. The gel was incubated in Tris and calcium chloride and stained with Coomassie blue. Clear zones in the gel indicate regions of proteolytic activity. When required, the protease inhibitor 1,10-phenanthroline was added to the wash and incubation buffers, and to the stained gel. For hyaluronidase activity, the hyaluronic acid was used as a substrate in SDS-PAGE following Miura et al. . Residual proteins (which may interfere with gel staining) were removed by adding S. griseus protease and incubating. The gel was stained with Alcian blue and the caseinolytic activity was also measured. An aliquot of casein (1%, in potassium phosphate buffer) substrate was incubated for 3 h in the absence (as blank) or the presence of jellyfish venom. The reaction was stopped by the addition of trichloroacetic acid and the casein hydrolysis was measured by modifying the Folin-Ciocalteu method. For the blank, the same amount of venom was added before the measurement. A standard graph was generated using standard tyrosine solution. The developed colors of reaction mixtures and standard mixtures were read at 660 nm. For the inhibition study of caseinolytic activity, metalloproteinase inhibitors (EDTA, EGTA, 1,10-penanthroline) and a serine proteinase inhibitor (PMSF) were used.
The venoms were preincubated with an inhibitor, and then its caseinolytic activity was measured as previously described. One unit of the caseinolytic activity was defined as the amount of enzyme, which hydrolyzed casein to liberate 1 mg of tyrosine per minute. Li et al.  studied the protease activity of venom from Rhopilema esculentum using the Folin-phenol of the Bakhtiar method . Briefly, casein in sodium phosphate buffer (PBS) and crude protein were preincubated. Then unhydrolyzed protein was precipitated with trichloroacetic acid and softly shaken. After centrifugation, Na2CO3 was added to the supernatant followed by the Folin-phenol reagent and then immediately shaken up. Absorbance at 640 nm was measured for the reaction mixture. One unit of protease activity was defined as 1 ng tyrosine released from casein hydrolyzed by protease of 1 mL of crude protein at 37 °C, pH 8.0, for 1 min. The effects of temperature, pH and additives on protease activity were also assessed.
2.4.2. Hemolytic Assay
The hemolytic assay is commonly used to determine if the protein solution has toxic properties such as the hemolysis of cells [12,13,20,24,36,40,41,55]. Yu et al.  studied the hemolytic activity of the Rhopilema esculentum venom according to the method previously described for C. marsupialis . Briefly, 0.5 ml of a 0.05% suspension of erythrocytes in Krebs Ringer phosphate buffer (KRP), pH 7.4, was incubated at 37 °C for 30 min with different amounts of venom. After centrifugation, the hemolytic activity was evaluated spectrophotometrically at 415 nm by assaying the hemoglobin released in the supernatant. Reference samples were employed using hypotonic lysis with water as a 100% lysis reference and the supernatant of 0.05% erythrocyte suspension (0.5 mL) incubated with 4.5 mL KRP at 37 °C for 30 min as the 0% reference. The HU50 was defined as the amount of venom required to cause 50% lysis. Junior et al.  also made a hemolytic assay slightly modified from the previous: human blood, freshly collected with heparin, was centrifuged to remove the buffy coat, and the erythrocytes obtained were washed three times in 0.85% saline and stored at 4 °C. Toxins at desired concentrations were added in the first well to erythrocyte buffer, and then were serially diluted in a two-fold ratio. Red blood cells in erythrocyte buffer were added to the toxins, and hemolysis was monitored by measuring attenuance at 630 nm for 20 min at room temperature. The final volume was 200 μL per well. The hemolysis percentage was determined at the end of the assay.
2.4.3. Antimicrobial Assay
Ovchinnikova et al.  developed an antimicrobial assay to assess venom toxicity activity. Antimicrobial activities of peptides were measured in radial diffusion assays by the agarose gel overlay technique according to Lehrer et al. . Samples were tested against E. coli and Listeria monocytogenes. The microorganisms were precultured in tryptic soy broth at 37 °C for 16 h. Aliquots of the bacteria-containing medium were transferred into a freshly prepared medium and incubated at 37 °C for 2.5 h to obtain mid-logarithmic phase microorganisms. Cell counts for each microorganism were determined, using a spectrophotometer, by the turbidity of cell suspensions at 620 nm. Suspension aliquots were mixed with sterile agarose solution in sodium phosphate buffer and poured into sterile plastic 90 mm Petri dishes. Wells made by a 3 mm applicator were filled with 5 µL test samples. The diameter of the inhibited growth zone (the microbe-free zone around a well) was measured assuming 0.1 mm as one unit of antimicrobial activity and subtracting 30 such units (the well diameter) from each result.
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