Nodularin

Manufactured by Enzo Life Sciences

Sourced in United States, Switzerland

Nodularin is a laboratory standard used for testing and research purposes. It is a cyclic pentapeptide derived from the cyanobacterium Nodularia spumigena. Nodularin serves as a reference compound for analytical and biochemical applications.

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13 protocols using nodularin

Immunofluorescence Analysis of Lamin Phosphorylation

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Cells were grown on uncoated glass coverslips under the standard
culture condition (see Cell Culture). Cells were fixed in
PHEM buffer (60 mM PIPES-KOH pH7.5, 25 mM HEPES-KOH pH7.5, 10 mM EGTA, 4 mM
MgSO₄) containing 4% formaldehyde, 0.5% Triton, and 100 nM
phosphatase inhibitor Nodularin (Enzo ALX-350–061) for 10 min at
37°C. Cells on coverslips were blocked with Blocking buffer (1% skim
milk and 5% goat serum in PBS), and then incubated with primary antibodies
in Blocking buffer. Antibodies used in immunofluorescence are: Alexa
647-conjugated rabbit monoclonal anti-phospho-Ser22-Lamin A/C antibody D2B2E
(labeled at Cell Signaling, product ID 97262BC, Lot 1, 1:100 dilution),
mouse monoclonal anti-pan-N-terminal-Lamin A/C antibody E1 (Santa Cruz
Biotechnology sc-376248, Lot # H2812, 1:5000), or mouse monoclonal
anti-full-length-Lamin A/C antibody 4C4 (Abcam ab190380, Lot
GR201137–1; 1:1000). Cells were incubated with secondary antibodies,
counterstained by DAPI, and cured in ProLong Gold mounting medium (Molecular
Probes, P36930). Cells were imaged using a Leica SP8 confocal microscope
with a 63x or 100x objective. See Quantification and Statistical Analysis for downstream
analyses.

Ikegami K., Secchia S., Almakki O., Lieb J.D, & Moskowitz I.P. (2020). Phosphorylated Lamin A/C in the nuclear interior binds active enhancers associated with abnormal transcription in progeria. Developmental cell, 52(6), 699-713.e11.

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Quantifying Microcystins in Ambient Waters

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Water, acetonitrile, methanol, and formic acid were all Optima LC/MS grade solvents and purchased from Fisher Scientific (Tewksbury, MA, USA). MCs MC-LR, RR, YR, WR, HtyR, HilR, D-Asp³-RR, D-Asp³-LR, LA, LF, LY, LW, and Nodularin were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). The surrogate C₂D₅ MC-LR was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). The Microcystin-Adda ELISA kit was purchased from Abraxis, Inc. (Warminster, PA, USA).
Standards were prepared in methanol and diluted to ng/L concentrations in LC MS grade water. All water samples were collected according to US EPA method 546: Determination of Total Microcystins and Nodularins in Drinking Water and Ambient Water by Adda Enzyme-Linked Immunosorbent Assay. Briefly, for ambient waters, EPA Method 546 does not require any preservatives to be added to the sample collection containers [12 ]. A 40 mL lake water sample was collected in PETG bottle at approximately 30 cm below the surface of the water. Samples were stored on ice after collection and until arrival to the lab where they were then frozen at −8 °C. MCs were released from the cells by subjecting the samples to a series of three freeze thaw cycles and debris was removed by filtering through a 1 µm glass fiber filter [18 (link),21 (link),23 (link)].

Birbeck J.A., Westrick J.A., O’Neill G.M., Spies B, & Szlag D.C. (2019). Comparative Analysis of Microcystin Prevalence in Michigan Lakes by Online Concentration LC/MS/MS and ELISA. Toxins, 11(1), 13.

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Quantification of Cyanotoxins and Analogues

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Standards of MC-LR, -LA, -LW, -LF, -LY, -RR, -YR, -WR, [D-Asp3]MC-LR, [D-Asp3]MC-RR, and MC-HilR isolated from Microcystis aeruginosa (analytical standard grade, ≥95% HPLC) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Anatoxin-a (synthetic, ≥98% TLC), CYN (isolated from Cylindrospermopsis raciborskii, ≥95% HPLC), nodularin (isolated from Nodularia spumigena, ≥95% HPLC) were also purchased from Enzo Life Sciences. Aeruginosamides B and C, anabaenopeptins B and F, cyanopeptolin 1040 MB, microginin 690, micropeptin 1106, and oscillamide Y (synthetic, HPLC grade, purity ≥95%) were purchased from LKT Labs (St. Paul, Minnesota, US). L-BMAA hydrochloride (≥97% NMR) was purchased from Sigma Aldrich (St. Louis, Missouri, US). Lyngbyatoxin-a was obtained in kind from Drs. Patrick Videau and Benjamin Philmus (Oregon State University, Department of Pharmaceutical Sciences) and prepared as previously described (Videau et al., 2016 (link)). All standards were purchased or obtained as dried powder and dissolved in 100 μl dimethyl sulfoxide (DMS0). Stock solutions were prepared in 0.1 mg/mL solution and stored for no longer than two weeks. Dilutions (0.5% DMSO) were made at 5000, 500, 50, 5, and 0.5 μg/L to represent a range of realistic bloom scenario concentrations.

Roegner A., Truong L., Weirich C., Schirmer M.P., Brena B., Miller T.R, & Tanguay R. (2019). Combined Danio rerio embryo morbidity, mortality and photomotor response assay: a tool for developmental risk assessment from chronic cyanoHAB exposure. The Science of the total environment, 697, 134210.

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Quantification of Cyanotoxins in Aquatic Samples

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Lyophilized analytical standards of microcystin-LA, LF, LR, LW, LY, RR, YR, and nodularin, all with manufacturer reported purities of ≥95%, were purchased from Enzo Life Sciences, Inc. (Framingdale, New York, USA). Stock solutions were prepared at concentrations of 10 μg/mL in methanol. Certified reference materials (CRMs) of microcystin-LR, RR, and nodularin, in methanol, as well as anatoxin-a, cylindrospermopsin, and saxitoxin were purchased from the National Research Council Canada (Ottawa, Ontario, Canada). All MC standards were stored in the dark at −20 °C. All other standards were stored according to the manufacturer's recommendations. For LC-MS/MS and PPIA analyses, Optima LC/MS Grade acetonitrile, methanol, and water were purchased from Fisher Scientific (Pittsburgh, PA, USA) and used for sample preparation and LC eluents. Trifluoroacetic acid (Chromasolv^® for HPLC, >99.0%) and Tween 20 for PPIA extractions were purchased from Sigma Aldrich (St. Louis, MO, USA).

Marsan D.W., Conrad S.M., Stutts W.L., Parker C.H, & Deeds J.R. (2018). Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit. Heliyon, 4(3), e00573.

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Quantitative Analysis of Cyanotoxin Standards

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Mobile phases were prepared from LC-MS-grade acetonitrile (Fisher Optima, ThermoFisher, Greater London, UK) and water used for LC-MS was obtained in-house. Sample preparation reagents were HPLC grade. Toxin standards used for preparation of calibration solutions (MC-RR, MC-LA, MC-LY, MC-LF, MC-LW, MC-YR, MC-WR, MC-HilR, MC-HtyR, MC-LR, [Asp3] MC-LR and Nodularin) were all obtained from Enzo Life Sciences, Exeter, UK. A certified standard of [Dha7] MC-LR was obtained from the Institute of Biotoxin Metrology, National Research Council Canada (NRCC, Halifax, NS, Canada). Reference standards received as solid films were dissolved in 50% aqueous methanol, to form stock solutions. A mixed stock solution was subsequently prepared by combining aliquots of each stock, followed by further dilutions to create seven-level suite of working calibration standards between 0.33 ng/mL to 327 ng/mL per toxin.

Turner A.D., Dhanji-Rapkova M., O’Neill A., Coates L., Lewis A, & Lewis K. (2018). Analysis of Microcystins in Cyanobacterial Blooms from Freshwater Bodies in England. Toxins, 10(1), 39.

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Analytical Standards for Cyanotoxin Detection

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Water, acetonitrile, methanol, acetic acid, and formic acid were all Optima LC/MS grade solvents and purchased from Fisher Scientific (Tewksbury, MA, USA). Microcystin standards D-Asp³-RR, MC-RR, YR, HtyR, LR, D-Asp³-LR, HilR, WR, LA, LY, LW, and LF, as well as nodularin and cylindrospermopsin were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Microcystin Dha⁷-LR was purchased from National Research Council Canada (Ottawa, ON, Canada). Microcystin Leu¹-LR was purchased from GreenWater Laboratories (Palatka, FL, USA). D-Asp³-E-Dhb⁷-RR was purchased from Sigma Aldrich. Anabaenopeptin A and B, oscillamide Y, oscillaginin A, microginin 690 methyl ester, aeruginosamide B and C, cyanopeptolin 1040 MB and 1007, and micropeptin 1106 were purchased from MARBIONC (Wilmington, NC, USA). Anabaenopeptin F was synthetically created by the Stockdill laboratory at Wayne State University (Detroit, MI, USA). ¹⁵N MC-LR was purchased from Cambridge Isotope Laboratories, INC. (Tewksbury, MA, USA).

Shahmohamadloo R.S., Rudman S.M., Clare C.I., Westrick J.A., Wang X., De Meester L, & Fryxell J.M. (2023). Intraspecific genetic variation is critical to robust toxicological predictions of aquatic contaminants. bioRxiv.

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Microcystin quantification using LC-MS and ELISA

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Water, acetonitrile, methanol, acetic acid, and formic acid were all Optima LC–MS grade solvents purchased from Fisher Scientific (Tewksbury, MA, USA). Additionally, 2-mercaptoethanol was purchased from Sigma Aldrich (St. Louis, MO, USA). MCs MC-LR, RR, YR, WR, HtyR, HilR, [d-Asp³]-RR, [d-Asp³]-LR, LA, LF, LY, and LW, as well as nodularin, were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). The surrogate C₂D₅ MC-LR was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). The Microcystins-Adda ELISA kit was purchased from Abraxis, Inc. (Warminster, PA, USA). Standards were prepared in methanol and diluted in LC–MS grade water. Samples were stored on ice after collection and until arrival to the laboratory where they were then frozen at −4 °C. Samples then went through a series of three freeze–thaw cycles before analysis [31 (link),39 (link),40 (link)].

Birbeck J.A., Peraino N.J., O’Neill G.M., Coady J, & Westrick J.A. (2019). Dhb Microcystins Discovered in USA Using an Online Concentration LC–MS/MS Platform. Toxins, 11(11), 653.

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Protein Phosphatase 1 Inhibition Assay

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Assays for the inhibition of Protein phosphatase 1 were based on described methods [64 (link)]. Protein phosphatase 1 (1.7 U·mL⁻¹, New England Biolabs, Ipswich, MA, USA) was dissolved in buffer solution containing 50 mM Tris (Sigma-Aldrich, St. Louis, MO, USA, pH 7.4), 20 mg·mL⁻¹ BSA (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM MnCl₂ × 4 H₂O (Sigma-Aldrich, St. Louis, MO, USA) and 0.2 mM DTT (Sigma-Aldrich, St. Louis, MO, USA). The substrate 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma-Aldrich, St. Louis, MO, USA) was dissolved (15 mM) in buffer solution with 0.5 M Tris (pH 8.1), 20 mM MgCl₂ × 6 H₂O (Riedel-De-Haën, Seelze, Germany), 0.2 mM MnCl₂ × 4 H₂O and 1 mg·mL⁻¹ BSA. Inhibitor (10 µL) or fraction dilution (10 µL), enzyme (25 µL) and substrate (200 µL) were added to the wells. The plate was incubated at 37 °C and the absorbance at 405 nm was read after 120 min. The inhibitor nodularin (Enzo Life Sciences, Lausen, Switzerland) was diluted in ultrapure water and six concentrations (0.125, 0.25, 0.5, 1, 2, and 4 ng·mL⁻¹) were used as positive control.

Häggqvist K., Toruńska-Sitarz A., Błaszczyk A., Mazur-Marzec H, & Meriluoto J. (2016). Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae) with Bioactive Properties. Toxins, 8(4), 108.

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Selectivity Assay for Monoclonal Antibodies

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Indirect cELISAs were completed using a panel of inhibitors to determine the selectivity of the mAbs. The cELISA procedure was nearly the same as that described for the serum screening, except for the addition of inhibitors (50 µL), which were mixed with 50 µL of the antibody solution during the primary antibody incubation step. The inhibitors tested were α-AMA (≥90%, Enzo Life Sciences, Farmingdale, NY, USA), β-AMA (≥90%, Enzo), γ-AMA (≥90%, Enzo), microcystin-LR (≥95%, Enzo), nodularin (≥95%, Enzo), phalloidin (>90%, Enzo), phallacidin (≥85%, Sigma), pysilocybin (>99%, Cerilliant, Round Rock, TX, USA), muscimol (>99%, Abcam, Cambridge, MA, USA), ibotenic acid (>98%, Abcam). Each analyte stock was dissolved in dH₂O, then serially diluted into TBST, starting at the highest concentration of ,000 ng mL⁻¹, and assessed in triplicate. Data were analyzed using a 4-parameter logistic equation (GraphPad Prism 7 Software, La Jolla, CA, USA) to determine the concentration of inhibition at half of the maximal signal (IC₅₀). Cross-reactivity (%) was calculated as follows: (IC₅₀ α-AMA)/(IC₅₀ test inhibitor) × 100.

Bever C.S., Hnasko R.M., Cheng L.W, & Stanker L.H. (2019). A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins. Toxins, 11(12), 724.

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Purification and Characterization of Toxins

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The toxins, α-AMA (≥90%), β-AMA (≥90%), γ-AMA (≥90%), microcystin-LR (≥95%), nodularin (≥95%), and phalloidin (>90%), were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Phallacidin (≥85%), sodium periodate (NaIO₄, >99.8%), sodium borohydride (NaBH₄, 99%), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol, Tween-20, keyhole limpet hemocyanin (KLH), and SuperSignal West Pico PLUS chemiluminescent substrate were purchased from Fisher Scientific (Waltham, MA, USA). Secondary antibody goat-anti-rabbit-horseradish peroxidase conjugate was purchased from Abcam (Cambridge, MA, USA). The hapten OSu-Glu-vc-PAB-C6-amanitin (LB) was purchased from Levena Biopharma (San Diego, CA, USA).

Bever C.S., Barnych B., Hnasko R., Cheng L.W, & Stanker L.H. (2018). A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin. Toxins, 10(7), 265.

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