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Microcystin

Microcystins are a class of cyclic heptapeptide hepatotoxins produced by certain cyanobacteria species.
These potent liver toxins can contaminate freshwater supplies and pose a serious threat to human and animal health.
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Most cited protocols related to «Microcystin»

1
Cocaine Alters mGluR1/5 Protein Expression
Cited 13 times
Our first immunoblotting study was aimed to investigate the influence of a history of cocaine self-administration experience upon the protein expression of mGluR1/5 within PFC subregions at early versus protracted withdrawal. For this study, within each cohort of rats, separate groups of Sal1h, Sal6h and Coc6h rats underwent a 2-h cue test session under extinction conditions at either 3 or 30 days withdrawal (n=12 per group at the start of the experiment) to assay for cue-reinforced lever-pressing behavior. Immediately upon completion of this cue test, animals were killed by rapid decapitation. The dmPFC (anterior cingulate and dorsal prelimbic cortices) and vmPFC (ventral prelimbic and infralimbic cortices) were dissected out over ice in a manner identical to that described by others (Ghasemzadeh et al., 2009 (link)). The tissue derived from animals in this study was immunoblotted in 2 separate ways. First, comparisons were made across the tissue from the 3 different self-administration groups separately for each time-point (i.e., Sal1h, Sal6h & Coc6h rats sacrificed at 3 days withdrawal were compared in one assay, and a parallel assay compared Sal1h, Sal6h & Coc6h rats sacrificed at 30 days withdrawal in a separate assay). This “single time-point” analysis was done to enable a comparison of protein expression between the SAL1h and SAL6h rats at each time-point, as well as to determine whether or not a history of cocaine self-administration influenced protein expression relative to a history of saline self-administration at that particular withdrawal time-point. For these assays, the immunoreactivity of the mGluR1 and mGluR5 bands from the Sal6h and Coc6h animals was normalized to the average of that for the Sal1h animals run on the corresponding gel (n=4–5/gel) and the data expressed as a percentage of that averaged Sal1h signal for each gel. The data for the 3-day and for the 30-day assays were analyzed separately using an univariate analysis of variance (ANOVA) across the 3 different self-administration groups to determine the presence/absence of a cocaine effect.
The next analysis examined for proteomic correlates of the increased lever-pressing exhibited by both saline and cocaine self-administering animals at 30 days of withdrawal, by comparing tissue from the Sal6h and Coc6h animals sacrificed at both withdrawal time-points on the same gel. For this “time-course” assay, the immunoreactivity of the mGluR1 and mGluR5 bands were normalized to the average of the Sal6h-3 day withdrawal group run on the corresponding gel (n=3–4/gel) and the data expressed as a percentage of that averaged signal for each gel. The data were analyzed using orthogonal comparisons, within each self-administration group, to determine the presence/absence of time-dependent changes in protein expression. A second, follow-up, study assayed vmPFC and dmPFC tissue from Sal1h and Coc6h rats, sacrificed at 3 versus 30 days withdrawal, in the absence of any cue testing. This study was conducted to determine the extent to which our cocaine-induced changes in mGluR1/5 protein expression reflected alterations in the basal expression pattern of these receptors or some interaction between cocaine experience and the cue-reinforced testing conditions of our animals. The data analysis for this study was identical to that employed for the “time-course” study above.
The immunblotting procedures for detection of total mGluR1 and mGluR5 levels in brain tissue homogenate were identical to those described recently by our group (e.g., Cozzoli et al., 2009 (link), 2012 ; Goulding et al., 2011 (link)). In brief, tissue was homogenized in a solution consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 1 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, 1 mM orthovanadate, and 2 μM microcystin LR were included to inhibit phosphotases. After centrifugation at 10,000 g for 20 min, the supernatant of the homogenates were quantified for protein content using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) and stored at −80 °C. Protein samples (20 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis on Tris-Acetate gradient gels (3–8%) (Invitrogen, Carlsbad, CA). Wet polyvinylidene difluoride (PVDF) (Bio-Rad) membrane transfer was employed, and membranes were pre-blocked with either phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dried milk powder for a minimum of 2 hrs before overnight incubation with primary antibody. An anti-mGluR5 rabbit polyclonal antibody (Millipore, Billerica, MA; 1:1000 dilution) and an anti-mGluR1a mouse polyclonal antibody (Millipore; 1:500 dilution) were used for receptor detection. A rabbit primary anti-calnexin antibody (Stressgen, Ann Arbor, MI; 1:1000 dilution) was used as a control to ensure even protein loading and transfer. Membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Millipore; 1:5,000–1:10,000 dilution) or anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA; 1:10,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). Image J (NIH, Bethesda, MD) was used to quantify immunoreactivity levels.
Ben-Shahar O., Sacramento A.D., Miller B.W., Webb S.M., Wroten M.G., Silva H.E., Caruana A.L., Gordon E.J., Ploense K.L., Ditzhazy J., Kippin T.E, & Szumlinski K.K. (2013). Deficits in Ventromedial Prefrontal Cortex Group 1 Metabotropic Glutamate Receptor Function Mediate Resistance to Extinction during Protracted Withdrawal from an Extensive History of Cocaine Self-Administration. The Journal of Neuroscience, 33(2), 495-506a.
Publication 2013
2
Synaptoneurosomal Preparation and Western Blot Analysis
Cited 10 times
Synaptoneurosomal preparation was carried out as previously described25 (link). Briefly, dorsal hippocampi were rapidly dissected in cold dissection buffer (2.6 mM KCl, 1.23 mM Sodium Phosphate Monobasic, 26 mM Sodium Bicarbonate, 5 mM Kynurenic acid, 212 mM Sucrose, 10 mM Dextrose, 0.5 mM CaCl2, 1 mM MgCl2) followed by homogenization in 10 mM HEPES, 2 mM EDTA, 2 mM EGTA, 0.5 mM DTT, with phosphatase and protease inhibitor cocktails (Sigma Aldrich) using glass–teflon homogenizer. Homogenates were filtered through 100 μm nylon mesh filter and 5 μm nitrocellulose filters sequentially. Synaptoneurosomes were obtained by centrifugating the filtrate at 1000g for 10 minutes. The pellet was resuspended in the homogenization buffer.
Western blot analysis was done as previously reported25 (link). Specifically, hippocampal total extracts from rat were obtained by polytron homogenization in cold lysis buffer with protease and phosphatase inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 2 mM NaF, 2 mM Na4P2O7, 4U/ml aprotonin, 2mM DTT, 1 mM EGTA, 1μM microcystin, 1mM benzamidine). Protein concentrations were determined using the BioRad protein assay (BioRad Laboratories, Hercules, CA). Equal amounts of total protein (10–20 μg/lane) were resolved on denaturing SDS–PAGE gels and transferred to Hybond–P membranes (Millipore) by electroblotting. Membranes were dried and then reactivated in methanol for 5 minutes and then washed with 3 changes of water. The membrane was then blocked in 3% milk/TBS or according to manufacturers’ instruction for 1 hour at room temperature, then incubated with primary antibody overnight at 4°C in solution per manufacturers’ suggestion. A full–length western blot image for each antibody used in this study is shown in Supplementary Fig. 8. All antibodies had been previously used and tested for specificity, as specified in the legend of supplementary Fig.8. Antibodies: anti–pCREB (1/1000) (Cat # 06-519), anti–GluA1 (1/2000) (Cat # AB-1504), anti–CaMKIIα (1/2000) (Cat # 05-532), anti–PLCγ (1/1000) (Cat # 05-163), anti–Synapsin–1 (1/2000) (Cat # AB-1543P) were purchased from Millipore (Billerica, MA). Anti–CREB (1/1000) (Cat # 9197), anti–pCaMKIIα (1/5000) (Cat # 3361s), anti–ERK1/2 (1/2000) (Cat # 9102), anti–pERK1/2 (1/2000) (Cat # 9101s), anti–Akt (1/1000) (Cat # 4691s), anti–TrkB (1/1000) (Cat # 4603s) and anti–pAkt (1/1000) (Cat # 4060s) and anti–pMSK1 (1/1000) (Cat # 9595P) were purchased from Cell Signaling Technology (Danvers, MA), anti–MSK1 (1/1000) (Cat # AF2518) was purchased from R&D systems, anti–Arc (1/1000) (Cat # 156003) was purchased from Synaptic Systems (Goettingen, Germany), anti–pTrkB (1/1000) (Cat # 2149-1) was purchased from Epitomics (Burlingame, CA), anti–pPLCγ (1/1000) (Cat #700044) was purchased from Invitrogen (Carlsbad, CA), and anti–pSynapsin–1 (Cat #S8192) was purchased from Sigma (St. Louis, MO) anti–Zif268 (egr–1; 1/500) (Cat #Sc-101), and anti–actin–HRP (1/4000) (Cat #Sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The membranes were washed, treated with secondary HRP–labeled donkey anti–rabbit or –goat anti–mouse antibody (1/4000, GE Healthcare, Waukesha, WI) for 1 hour, washed again and incubated with HRP–streptavidin complex and ECL detection reagents (GE healthcare, Waukesha, WI). Membranes were exposed to Denville Scientific HyBlotCL (Denville Scientific, Metuchen, NJ) and quantitative densitometric analysis was performed using NIH ImageJ. Changes were quantified within gels, and an n=3-4 rats per group in every gel were used. Actin was used as loading control for all markers. All membranes on which the specific markers were investigated were stripped with stripping buffer (1% SDS, 31.25 mM Tris HCl pH 6.8, 0.7% β–mercaptoethanol) and probed with actin. In cases where phospho–markers were probed, the same membranes, whenever possible, were stripped again with stripping buffer after actin levels have been assessed and probed for their respective total levels. The experiments were designed as follows: First we investigated the effect of RU486 or anti–BDNF on changes elicited by training. Hence, the expression levels of the markers in trained rats treated either with vehicle, RU486 or anti–BDNF compared to naïve treated with vehicle were determined. Subsequently, having found an effect on trained rats, we determined the effect of RU486 or anti–BDNF on naïve animals. As these experiments were run separately they are shown separately in the relative figures. Examples of full-length blots are shown in Supplementary Figures 8-17.
Chen D.Y., Bambah–Mukku D., Pollonini G, & Alberini C.M. (2012). Glucocorticoid receptors recruit the CaMKIIα, BDNF–CREB pathways to mediate memory consolidation. Nature neuroscience, 15(12), 1707-1714.
Publication 2012
3
Alcohol Drinking Regulates Mesocorticolimbic mGluR-Homer-PI3K
Cited 10 times
Chronic, continuous alcohol consumption elevates NAC levels of Homer2, and associated glutamate receptor proteins (Szumlinski et al., 2008b (link)). To determine whether or not alcohol drinking up-regulates the mesocorticolimbic expression of members of the mGluR-Homer-PI3K signaling pathway in the fully extended SHAC drinking model (for discussion, Finn et al., 2005 (link)), B6 mice were subjected to 6 bouts of SHAC drinking over an 18-day period with 5% alcohol available for 30 min, every 3rd day (see above). Control animals received tap water in an identical 50 ml sipper tube during each of the 30-min sessions. Animals were decapitated 24 hrs following the 6th alcohol presentation, brains were sectioned (1.0 mm thick) along the coronal plane and the entire prefrontal cortex, NAC, dorsal striatum and hippocampus were dissected out over ice. As the mGluR5F1128R mutation reduced binge drinking with the SHAC procedure (see Results), a second experiment assessed genotypic differences in basal NAC protein expression in experimentally naïve WT and mGluR5F1126R mutant mice. Finally, a 3rd immunoblotting experiment was conducted on NAC tissue from selectively bred SHAC and SLAC mice (see above) to further relate genetic vulnerability in binge alcohol drinking to mGluR/Homer/PI3K expression in the NAC (i.e., was mGluR5/Homer/PI3K expression in the NAC a correlated response to selection for binge drinking?). For this experiment, frozen whole brains from S4 SHAC and SLAC offspring 3 months following alcohol testing were sectioned along the coronal plane (1 mm thick) at the level of the NAC and the entire NAC and dorsal striatum dissected out over ice.
As described in recent reports by our group (Ary and Szumlinski, 2007 (link); Ary et al., 2007 (link); Szumlinski et al., 2008b (link)), the tissue from all the experiments outlined above was homogenized in a medium consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 50 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, and 2 μM microcystin LR were included to inhibit phosphatases. Samples were then subjected to low-speed centrifugation at 10,000 g for 20 min. Protein determinations were performed using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA), according to the manufacturer's instructions and homogenates were stored at -80°C until immunoblotting was completed.
For immunoblotting, protein samples (5-20 μg) were subjected to a SDS-polyacrylamide gel electrophoresis. Bis-Tris gradient gels (4-12%) (Invitrogen) were used for separation of Homers, PI3K, and the p(Tyr)p85α PI3K binding motif, the latter of which was employed to index PI3K activity (e.g., Zhang et al., 2006 (link)). Tris-Acetate gradient gels (3-8%) (Invitrogen) were used for separation of Homers, as well as the glutamate receptor proteins. Proteins were transferred to PVDF membranes, preblocked with phosphate-buffered saline containing 0.1% (v/v) Tween-20 and either 5% (w/v) bovine serum albumin [for p(Tyr)p85α PI3K binding motif] or 5% (w/v) nonfat dried milk powder (for all other proteins) for no less than 1 hr before overnight incubation with primary antibodies. The following rabbit polyclonal antibodies were used: anti-Homer 2a/b and anti-Homer 1b/c (Dr. Paul F Worley, Johns Hopkins University School of Medicine; 1:1000 dilution), anti-mGluR5 (Upstate Cell Signaling Solutions, Lake Placid, NY; 1:1000 dilution), anti-NR2a and anti-NR2b (Calbiochem, San Diego, CA; 1:1000 dilution), anti-PI3K antibody (Upstate, Lake Placid, NY; 1:1000 dilution), and anti-p-(Tyr) PI3K p85α binding motif (Cell Signaling Technology, Beverly, MA; 1:500 dilution). An anti-mGluR1a mouse polyclonal antibody (Upstate, Lake Placid, NY; 1:1000 dilution) was also used. Membranes were washed, incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary anti-body (Upstate, Charlottesville, VA; 1:20,000-1:40,000 dilution) or anti-mouse secondary anti-body (Jackson Immuno Research Laboratories, West Grove, PA; 1:20,000-1:40,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). A rabbit anti-calnexin polyclonal primary antibody (Stressgen, Victoria, BC) was also used to index protein loading and transfer. The levels of immunoreactivity for all proteins were quantified using Image J (NIH, Betheseda, MD) and the immunoreactivity for each protein of interest for each animal was first normalized to that of its appropriate calnexin signal to provide a protein/calnexin ratio. These ratios were then normalized to the mean ratios for each protein of the water or genetic control for each individual gel (n=3-4/gel).
Cozzoli D.K., Goulding S.P., Zhang P.W., Xiao B., Hu J.H., Ary A.W., Obara I., Rahn A., Abou-Ziab H., Tyrrel B., Marini C., Yoneyama N., Metten P., Snelling C., Dehoff M.H., Crabbe J.C., Finn D.A., Klugmann M., Worley P.F, & Szumlinski K.K. (2009). Binge Drinking Upregulates Accumbens mGluR5–Homer2–PI3K Signaling: Functional Implications for Alcoholism. The Journal of Neuroscience, 29(27), 8655-8668.
Publication 2009
4
Insulin Signaling Pathway Inhibition Assay
Cited 8 times
Except where indicated otherwise, cells were serum starved overnight and incubated with inhibitors or 0.1% DMSO for 30 min prior to stimulation with 100 nM insulin for 10 min. All inhibitors were either synthesized as previously described [21 (link),24 (link),55 (link)] or were from Calbiochem (rapamycin and Akti-1/2). Cells were lysed by scraping into ice cold lysis buffer followed by brief sonication. Lysates were cleared by centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies from Cell Signaling Technology. Unless otherwise indicated, cells were lysed in 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02% NaN3, 20 nM microcystin (Calbiochem), Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail, and 2 mM PMSF. For Figures 6A and 7A, and Figure S2A, cells were lysed in cap lysis buffer (140 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 4 mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail without EDTA and 2 mM PMSF).
Feldman M.E., Apsel B., Uotila A., Loewith R., Knight Z.A., Ruggero D, & Shokat K.M. (2009). Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2. PLoS Biology, 7(2), e1000038.
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Publication 2009
Antibodies Buffers Cells Centrifugation Cold Temperature Edetic Acid inhibitors Insulin Magnesium Chloride microcystin Nitrocellulose Nonidet P-40 Protease Inhibitors protein phosphatase inhibitor-1 SDS-PAGE Serum Sirolimus Sodium Azide Sodium Chloride Sulfoxide, Dimethyl Triton X-100 Tromethamine
5
Striatal Glutamate Signaling Regulates Alcohol Intake
Cited 8 times
Behavioral pharmacological and genetic studies implicate members of the mGluR-Homer-PI3K and -PKCε signaling cascades in regulating alcohol intake in rodents (Backstrom et al. 2004 (link); Besheer et al. 2008 (link); Blednov & Harris 2008 (link); Cozzoli et al. 2009 ; Hodge et al. 2006 (link); Lominac et al. 2006 ; Olive et al. 2005 (link); Schroeder et al. 2005 (link)) and a recent study suggests that mGluR5-mediated activation of extracellular signal-regulated kinase (ERK) within the NAC shell subregion is involved in cue-induced reinstatement of alcohol-seeking behavior (Schroeder et al. 2008 (link)). Thus, immunoblotting was conducted on tissue from both the dorsal and ventral aspects of the striatum to assess strain differences in basal and alcohol-induced changes in the expression of the constitutively expressed Homer proteins Homer1b/c and Homer2a/b, their associated Group1 metabotropic glutamate receptor (mGluR) subtypes mGluR1 and mGluR5 (e.g., Tu et al. 1999 (link)), as well as their associated NR2a/b subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor (Naisbitt et al. 1999 (link)). Moreover, we examined for strain differences in basal and alcohol-induced changes in the expression of the non-activated and activated forms of PKCε and ERK, as well as total PI3K expression and the levels of phospho-(Tyr)p85α as a read-out of PI3K activity (Zhang et al. 2006 (link)). Samples sizes ranged from 10–12 mice per group.
The immunoblotting procedures employed for the detection of glutamate receptors/Homers/kinases were identical to those previously described by our group (Cozzoli et al. 2009 ; Ary & Szumlinski 2007 (link); Ary et al. 2007 (link); Szumlinski et al. 2008 (link)). In brief, at 24 hrs withdrawal from repeated alcohol/saline treatment, the shell and core subregions of the NAC (ventral striatum) and/or the dorsal aspect of the striatum were dissected over ice. To extend early preliminary data (n=2) demonstrating significant and selective Homer2a/b knock-down in the NAC by our AAV-shRNA constructs, the NAC from control and shRNA-infused mice was dissected over ice at 2 months post-AAV infusion (see below). For both studies, brains were placed in an ice-cold mouse brain mold (Braintree Scientific, Braintree, MA), sectioned along the horizontal plane (0.5 mm thick) and micropunches of tissue made with an 18-gauge needle for shell/core and a 1 mm biopsy punch for the dorsal striatum. Tissue punches were then homogenized in a solution consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 50 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, and 2 μM microcystin LR was included to inhibit phosphatases. After centrifugation at 10,000 g for 20 min, the homogenates were quantified using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) and stored at −80 °C. Protein samples (20 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis and Tris-Acetate gradient gels (3–8%) (Invitrogen, Carlsbad, CA) were used to separate glutamate receptors, while Bis-Tris gradient gels (4–12%) (Invitrogen) or Tris-Acetate gels were employed to separate Homers and the kinases. Wet polyvinylidene difluoride (PVDF) (Bio-Rad) membrane transfer was employed, and membranes were pre-blocked with either phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dried milk powder (for non-phosphorylated proteins and kinases) or a 5% (w/v) bovine serum albumin phosphate-buffered saline solution (for phosphorylated kinases) for a minimum of 2 hrs before overnight incubation with primary antibody. The following rabbit polyclonal antibodies were used: anti-Homer 1b/c and anti-Homer 2a/b (Dr. Paul F Worley, Johns Hopkins University School of Medicine; 1:1000 dilution), anti-mGluR5 (Millipore, Billerica, MA; 1:1000 dilution), anti-NR2a and anti-NR2b (EMD Chemicals, Gibbstown, NJ; 1:1000 dilution), anti-PI3K (Millipore; 1:1000 dilution), anti-p(Tyr)p85alpha binding motif (Cell Signaling Technology, Beverly, MA; 1:500 dilution), anti-ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:2000 dilution), anti-PKCε (Santa Cruz Biotechnology; 1:1000 dilution), and anti-p(Ser729)PKCε (Santa Cruz Biotechnology; 1:500 dilution). The following mouse primary polyclonal antibodies were also used: anti-mGluR1 (Millipore; 1:500 dilution) and anti-p(Tyr204)ERK1/2 (Santa Cruz Biotechnology; 1:1000 dilution). A rabbit primary anti-calnexin antibody (Stressgen, Ann Arbor, MI; 1:1000 dilution) was used as a control to ensure even protein loading and transfer. Membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Millipore; 1:5,000–1:10,000 dilution) or anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA; 1:10,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). Primary antibodies were stripped off PVDF membranes with ReBlot Plus Strong Antibody Stripping Solution (Millipore). Image J (NIH, Bethesda, MD) was used to quantify immunoreactivity levels. An analysis of the immunoreactivity for calnexin indicated even protein loading and transfer (e.g., Figure 1). Thus, the density × area measurements for each protein band was averaged over the D2-saline control samples within a gel and all bands on that gel were expressed as percent of the average D2-saline value. To obtain an index of kinase activation, the density × area measurements for each phospho-protein was normalized to that of its corresponding non-phosphorylated protein prior to expressing the data as percent average D2-saline values.
Goulding S.P., Obara I., Lominac K.D., Gould A.T., Miller B.W., Klugmann M, & Szumlinski K.K. (2010). Accumbens Homer2-mediated signaling: A factor contributing to mouse strain differences in alcohol drinking?. Genes, brain, and behavior, 10(1), 111-126.
Publication 2010

Most recents protocols related to «Microcystin»

1
Microcystin-LR Quantification in Plants
MC-LR in plants was determined according to Jiang et al. [1 (link)]. Leaf tissues were ground to fine powder under liquid nitrogen, and then extracted with 5% acetic acid followed by 90% methanol. The extracts were pooled, centrifuged at 15,000 rpm for 20 min, and diluted with Milli-Q water before being applied to Oasis HLB cartridges (Waters, Ireland). The MC-LR in the cartridges was eluted with 90% methanol and evaporated to dryness. The dried exacts were reconstituted with Milli-Q water and analyzed by ELISA (Microcystin plate kit, Institute of Hydrobiology, Chinese Academy of Sciences). Extraction and centrifugation were conducted at 4–8 °C.
Jiang J., Shi Y., Tian F., Long T., Li X, & Ying R. (2024). Bioaccumulation of Microcystin-LR and Induced Physio-Biochemical Changes in Rice (Oryza sativa L.) at Vegetative Stage under Hydroponic Culture Conditions. Toxins, 16(2), 82.
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Publication 2024
2
Quantification of Cyanotoxin Production
Cultures grown on different N sources were collected at exponential growth phase for toxin analysis and particulate organic C (POC) measurement. Major toxin congeners (i.e. MC-LR, D-Asp MC-LR, and MC-HilR) were quantified using ultra-high performance liquid chromatography – mass spectrometry (UHPLC–MS) at University of Toledo following previously published protocols [46–48 (link)]. To compare MC production between cultures, POC concentrations of each culture were determined using a TOC analyzer (Shimadzu TOC-L equipped with an SSM-5000 solid state module, Shimadzu, Japan) to normalize the MC concentrations. Detailed procedures can be found in the Supplemental Material. Tukey’s HSD method was carried out to compare the effect of microbiomes and N sources.
Li W., Baliu-Rodriguez D., Premathilaka S.H., Thenuwara S.I., Kimbrel J.A., Samo T.J., Ramon C., Kiledal E.A., Rivera S.R., Kharbush J., Isailovic D., Weber P.K., Dick G.J, & Mayali X. (2024). Microbiome processing of organic nitrogen input supports growth and cyanotoxin production of Microcystis aeruginosa cultures. The ISME Journal, 18(1), wrae082.
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Publication 2024
3
Microcystin Quantification via ELISA
Samples of microcystins were evaluated as total and aqueous toxins using enzyme-linked immunosorbent assay (ELISA), following the manufacturer’s protocol (Eurofins Abraxis, Warminster, PA, USA). Samples of aqueous toxins were filtered through a 0.45 μm glass filter and frozen and samples of total toxins were frozen. Both aqueous and total samples were subjected to three freeze–thaw cycles, and total toxin samples were filtered through a 0.45 μm glass filter prior to analysis.
Lefler F.W., Barbosa M., Berthold D.E., Roten R., Bishop W.M, & Laughinghouse HD I.V. (2024). Microbial Community Response to Granular Peroxide-Based Algaecide Treatment of a Cyanobacterial Harmful Algal Bloom in Lake Okeechobee, Florida (USA). Toxins, 16(5), 206.
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Publication 2024
4
Arthrospira platensis Pigment Extraction
The dry biomass sample was prepared for extraction by weighing approximately 20 mg of Arthrospira platensis into a 2 mL plastic bottle with a cap (Eppendorf type). Then, 1.5 mL of 50% methanol (Lichrosolv, HPLC gradient grade, Merck KGaA, Darmstadt, Germany) was added to the sample, along with ultrapure water with a conductivity of 0.055 µS (v/v). The sample was subjected to ultrasonic extraction in a bath for 1 h, followed by centrifugation. The resulting extract (0.5 mL) was filtered using PTFE filters (0.45 µm, Thermo Fisher Scientific Inc., Waltham, MA, USA) prior to chromatography. Two parallel extractions were prepared.
Chromatographic determination was performed using an Agilent 1200 liquid chromatograph equipped with a diode array detector. The analytical column used was a Supelcosil ABZ + Plus column (150 mm × 4.6 mm, 5 µm, Supelco, Darmstadt, Germany), thermostated at 25 °C. A gradient of acetonitrile (Lichrosolv, HPLC gradient grade, Merck KGaA, Darmstadt, Germany) and ultrapure water (conductivity of 0.055 µS) was employed, with the addition of 0.1% trifluoroacetic acid (Trifluoroacetic acid, suitable for HPLC ≥ 99.0%, Merck KGaA, Darmstadt, Germany) to both solvents. The gradient profile included 20% acetonitrile at 0 min, which increased to 46% at 20 min, with a total chromatography time of 25 min at a flow rate of 1 mL min−1. Detection was performed at a wavelength of 238 nm.
Nikolova K., Gentscheva G., Gyurova D., Pavlova V., Dincheva I., Velikova M., Gerasimova A., Makedonski L, & Gergov G. (2024). Metabolomic Profile of Arthrospira platensis from a Bulgarian Bioreactor—A Potential Opportunity for Inclusion in Dietary Supplements. Life, 14(2), 174.
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Publication 2024
5
Quantifying Microcystins in CCA Serum
We used the microcystins (MCs) ELISA kit (502000; Cayman Chemical, MI, USA) in accordance with the manufacturer's instructions to measure the concentration of MC-LR in the serum of both CCA patients (n = 20) and healthy individuals (n = 10), in order to confirm the existence of MC-LR in CCA patients. In brief, 50 μL of both samples and microcystin (MC) standard solutions were carefully added to the designated test wells. Subsequently, microcystin-HRP tracer and microcystin ELISA monoclonal antibodies were then added to the wells. The subsequent phase comprised a precisely controlled incubation period of 2 h at 25 °C. After this incubation, an ELISA reader (Varioskan LUX, Thermo Scientific) was assayed to evaluate the absorbance at 450 nm. In addition, serum spiked with MCs was used as positive control.
Kongsintaweesuk S., Klungsaeng S., Intuyod K., Techasen A., Pairojkul C., Luvira V., Pinlaor S, & Pinlaor P. (2024). Microcystin-leucine arginine induces the proliferation of cholangiocytes and cholangiocarcinoma cells through the activation of the Wnt/β-catenin signaling pathway. Heliyon, 10(9), e30104.
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Publication 2024

Top products related to «Microcystin»

1
Microcystin lr by Enzo Life Sciences
Sourced in United States
Microcystin-LR is a reference standard for use in the analysis of microcystins, a group of cyclic heptapeptide hepatotoxins produced by certain cyanobacteria. It is used for method development, validation, and quantification in analytical testing.
2
Mc lr by Enzo Life Sciences
Sourced in United States, Switzerland, United Kingdom
MC-LR is a laboratory reference standard used for the analysis and detection of microcystins, a class of cyanotoxins, in environmental and biological samples. It serves as a calibration and quality control material for analytical methods employed in the monitoring and assessment of microcystin contamination.
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Fetal bovine serum (fbs) by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Australia, Japan, Canada, Italy, France, Switzerland, New Zealand, Brazil, Belgium, India, Spain, Israel, Austria, Poland, Ireland, Sweden, Macao, Netherlands, Denmark, Cameroon, Singapore, Portugal, Argentina, Holy See (Vatican City State), Morocco, Uruguay, Mexico, Thailand, Sao Tome and Principe, Hungary, Panama, Hong Kong, Norway, United Arab Emirates, Czechia, Russian Federation, Chile, Moldova, Republic of, Gabon, Palestine, State of, Saudi Arabia, Senegal
Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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Protease inhibitor cocktail by Roche
Sourced in United States, Switzerland, Germany, China, United Kingdom, France, Canada, Japan, Italy, Australia, Austria, Sweden, Spain, Cameroon, India, Macao, Belgium, Israel
Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.
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Trizol reagent by Thermo Fisher Scientific
Sourced in United States, China, Japan, Germany, United Kingdom, Canada, France, Italy, Australia, Spain, Switzerland, Netherlands, Belgium, Lithuania, Denmark, Singapore, New Zealand, India, Brazil, Argentina, Sweden, Norway, Austria, Poland, Finland, Israel, Hong Kong, Cameroon, Sao Tome and Principe, Macao, Taiwan, Province of China, Thailand
TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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Microcystin lr by Merck Group
Sourced in United States
Microcystin-LR is a laboratory reference standard used for the detection and quantification of microcystin-LR, a toxic substance produced by certain cyanobacteria. It serves as a calibration and verification tool for analytical methods employed in environmental monitoring and research.
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Protease inhibitor cocktail by Merck Group
Sourced in United States, Germany, China, United Kingdom, Italy, Japan, Sao Tome and Principe, France, Canada, Macao, Switzerland, Spain, Australia, Israel, Hungary, Ireland, Denmark, Brazil, Poland, India, Mexico, Senegal, Netherlands, Singapore
The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.
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Mc rr by Enzo Life Sciences
Sourced in Switzerland, United Kingdom, Germany, United States
MC-RR is a laboratory standard produced by Enzo Life Sciences. It is a microcystin variant that can be used for research and analytical purposes. This product is offered in a purified form for use in various applications.
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Formic acid by Merck Group
Sourced in Germany, United States, Italy, United Kingdom, France, Spain, China, Poland, India, Switzerland, Sao Tome and Principe, Belgium, Australia, Canada, Ireland, Macao, Hungary, Czechia, Netherlands, Portugal, Brazil, Singapore, Austria, Mexico, Chile, Sweden, Bulgaria, Denmark, Malaysia, Norway, New Zealand, Japan, Romania, Finland, Indonesia
Formic acid is a colorless, pungent-smelling liquid chemical compound. It is the simplest carboxylic acid, with the chemical formula HCOOH. Formic acid is widely used in various industrial and laboratory applications.
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Mc lf by Enzo Life Sciences
Sourced in United Kingdom, Switzerland, United States, Germany
The MC-LF is a laboratory instrument designed for the detection and quantification of microcystin-LF (MC-LF), a cyanotoxin commonly found in freshwater environments. The core function of this product is to provide accurate and reliable measurement of MC-LF levels, enabling researchers and environmental monitoring agencies to assess water quality and potential health risks.

More about "Microcystin"

Microcystins are a class of potent cyclic heptapeptide hepatotoxins produced by certain species of cyanobacteria (also known as blue-green algae).
These toxins can contaminate freshwater supplies and pose a serious threat to human and animal health.
MC-LR (Microcystin-LR) is one of the most commonly studied and toxic variants.
Researchers often utilize analytical techniques like HPLC, LC-MS, and ELISA to detect and quantify Microcystins in environmental and biological samples.
FBS (fetal bovine serum) and protease inhibitor cocktails may be used to help stabilize Microcystin samples during extraction and analysis.
TRIzol reagent is another common tool for RNA/DNA isolation from cyanobacteria cells.
Related Microcystin variants include MC-RR and MC-LF, which display similar toxicological profiles.
Formic acid is sometimes used in sample preparation to enhance Microcystin detection.
PubCompare.ai's AI-powered platform can help researchers optimize their Microcystin analysis protocols by identifying the most effective methods and products from the scientific literature, preprints, and patents.
By streamlining the research process, scientists can improve reproducibility and get better results in their Microcystin studies.