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Phospholipase c

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Phospholipase C is an enzyme that catalyzes the hydrolysis of phospholipids, specifically phosphatidylinositol (PI) and phosphatidylcholine (PC), into diacylglycerol (DAG) and the corresponding phosphate-containing head group. This enzymatic activity plays a crucial role in signal transduction pathways within cells.

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Lab products found in correlation

Glimepiride, by Merck Group (1 mentions) Lipofectamine plus, by Thermo Fisher Scientific (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) Wst 1 reagent, by Roche (1 mentions) Anti tubulin, by Merck Group (1 mentions) Fluoro jade b, by Merck Group (1 mentions) 96 well plate, by Greiner (1 mentions) Gs 800 calibrated, by Bio-Rad (1 mentions) F60 sonic dismembrator, by Thermo Fisher Scientific (1 mentions) Dnase, by Merck Group (1 mentions) Ricinoleic acid, by Merck Group (1 mentions) Phospholipase a2, by Merck Group (1 mentions) Trioleoyl glycerol, by Larodan (1 mentions)

Close spelling variants of this product

Phospholipase C from B. cerus Phospholipase C phosphatidylinositol-specific (PI-PLC) from Bacillus cereus Phosphatidylinositol-specific phospholipase C Phospholipase C from Bacillus cereus Phospholipase C from B. cereus

12 protocols using phospholipase c

1

Click-chemistry based cell assay

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We design our control experiments according to the click chemistry based assays reported in Ref. (10). NIH 3T3 cells cultured with 0.5 mM propargylcholine for 48 hours were fixed with 4 % PFA for 15 minutes, rinsed with 1 mL TBS buffer twice and incubated with 1 mL 1 mg/mL BSA in TBS buffer for 1 hour at 37 °C, with or without 0.02 U/mL phospholipase C (Type XIV from Clostridium perfringens, Sigma), in the presence of 10 mM CaCl2 (required for phospholipase C activity) (Supplementary Fig. 4b) or 10 mM EDTA (Supplementary Fig. 4c). The cells were then washed with TBS buffer and ready for SRS imaging.
Wei L., Hu F., Shen Y., Chen Z., Yu Y., Lin C.C., Wang M.C, & Min W. (2014). Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering. Nature methods, 11(4), 410-412.
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2

Click-chemistry based cell assay

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We design our control experiments according to the click chemistry based assays reported in Ref. (10). NIH 3T3 cells cultured with 0.5 mM propargylcholine for 48 hours were fixed with 4 % PFA for 15 minutes, rinsed with 1 mL TBS buffer twice and incubated with 1 mL 1 mg/mL BSA in TBS buffer for 1 hour at 37 °C, with or without 0.02 U/mL phospholipase C (Type XIV from Clostridium perfringens, Sigma), in the presence of 10 mM CaCl2 (required for phospholipase C activity) (Supplementary Fig. 4b) or 10 mM EDTA (Supplementary Fig. 4c). The cells were then washed with TBS buffer and ready for SRS imaging.
Wei L., Hu F., Shen Y., Chen Z., Yu Y., Lin C.C., Wang M.C, & Min W. (2014). Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering. Nature methods, 11(4), 410-412.
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3

Antibodies and Cell Reagents for Prion Research

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KA, glimepiride and phospholipase C were purchased from Sigma (Poole Dorset, UK). Fluoro-Jade B was from Millipore (Billerica, MA), SYBR green (Applied Biosystems, USA) and WST−1 reagents were from Roche (Basel, Switzerland). Lipofectamine plus was from Invitrogen (Carlsbad, CA). The following antibodies were used in this study: Anti-PrP SAF61 mouse monoclonal antibody (1:1000 diluted) antibody was from Spi-Bio (Cayman Chemical, Massy, France) and anti-PrP 6H4 mouse monoclonal antibody (1:5000 diluted western blotting and 1:250 immunocytochemistry) antibody was from Prionics (Schlieren, Switzerland). Mouse monoclonal antibody anti-tubulin (1:10000 diluted) was from Sigma (Poole Dorset, UK) and rabbit-raised polyclonal antibody against GFAP (1:500 diluted) was from Millipore (Billerica, MA).
Carulla P., Llorens F., Matamoros-Angles A., Aguilar-Calvo P., Espinosa J.C., Gavín R., Ferrer I., Legname G., Torres J.M, & del Río J.A. (2015). Involvement of PrPC in kainate-induced excitotoxicity in several mouse strains. Scientific Reports, 5, 11971.
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4

Quantifying Complement Regulation by DAF

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5×106 splenocytes from NP-KLH mice were incubated in HL-serum free media for 1h at 37°C, 5% CO2 in the presence or absence of 5ug/ml Phospholipase C (Sigma-Aldrich), washed with PBS 1%FCS and surface DAF expression quantified by flow cytometry. Complement regulatory capacity of DAF-TM was assessed in vitro by incubating 5×106 splenocytes from immunized animals in 20–40% WT or C3−/− mouse serum in Annexin V buffer with 1mM MgCl2 for 20min at 37°C, 5% CO2 and quantifying C3b deposition by flow cytometry.
Cumpelik A., Heja D., Hu Y., Varano G., Ordikhani F., Roberto M.P., He Z., Homann D., Lira S.A., Dominguez-Sola D, & Heeger P.S. (2021). Dynamic regulation of B cell Complement Signaling is Integral to Germinal Center Responses. Nature immunology, 22(6), 757-768.
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5

Lipoprotein Aggregation Measurement Assay

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Lipoprotein aggregation was measured using modified human low density lipoproteins (LDL) (21 (link)). Fifty μg of protein was mixed with an equal amount of LDL protein in a total volume of 200 μL reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, pH 7.4) in a 96-well plate (Greiner Bio-One). The reaction was started by addition of 200 mU phospholipase-C (Sigma-Aldrich) to the LDL apolipoprotein mixture, and the absorbance at 340 nm was recorded at 5 min intervals in a Thermo Varioskan plate reader for 2 h at 37 °C.
Sallee D.E., Horn J.V., Fuentes L.A, & Weers P.M. (2017). Expression of the C-terminal domain of human apolipoprotein A-I using a chimeric apolipoprotein. Protein expression and purification, 137, 13-19.
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6

Quantifying Clostridial Alpha Toxin Activity

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To determine the alpha toxin activity in the supernatants of strains JIR12058, EHE-NE18, CP56, JIR4866 and JIR4860, the lecithinase activity was assayed in an egg yolk agar well diffusion assay [30 (link),31 (link),32 (link)]. Therefore, small holes were pierced into Columbia agar supplemented with 2% egg yolk (v/v) with the rear end of a 20–200 µL pipette tip (diameter 7 mm), which were filled with 20 µL of supernatant per tested strain. A standard was included on each plate by preparing a two-fold dilution series of Phospholipase C (Sigma Aldrich) ranging from 1 to 0.0313 U/mL. Plates were incubated anaerobically overnight at 37 °C and scanned with a GS-800 calibrated densitometer (Bio-Rad Laboratories, Hercules, CA, USA). Alpha toxin activity was indicated by the development of turbidity. The area of the opaque zones was measured using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).
Hustá M., Ducatelle R., Van Immerseel F, & Goossens E. (2021). A Rapid and Simple Assay Correlates In Vitro NetB Activity with Clostridium perfringens Pathogenicity in Chickens. Microorganisms, 9(8), 1708.
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7

Apolipoprotein Binding to Lipoprotein Surface

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Binding of apoLp-III to lipoprotein surfaces was assessed using lipolyzed human low-density lipoprotein (LDL) as described earlier [30 (link)]. Fifty μg of LDL protein (Sigma Aldrich, Saint Louis, MO) was mixed with 100 mU phospholipase C (Sigma Aldrich, Saint Louis, MO) and incubated at 37 °C in the absence or presence of 200 μg of lipid-free or lipid-bound apoLp-III in Tris-HCl buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM CaCl2, pH 7.4). Incubations were carried out in a 96-well plate with a 200 μl for each sample in triplicate. LDL aggregation induced by phospholipase C was measured by the absorbance at 340 nm at every 5 min interval for 1 h.
Wijeratne T, & Weers P.M. (2019). Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein. Molecular and cellular biochemistry, 458(1-2), 61-70.
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8

Synthetic Pathway for Acetyl-TAG Lipids

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The TAG standards used in this study were synthesized from their respective phosphatidylcholine (PtdCho) orthologs (Avanti polar lipids, Alabaster, AL) using a two-step method. In the first step, PtdCho containing the desired sn-1/2 acyl composition were converted to 1,2-DAG using phospholipase C (Sigma-Aldrich, St. Louis, MO). Acetyl-TAG were subsequently synthesized by treating 1,2-DAG with acetic anhydride/pyridine (3:2) overnight at room temperature [9 (link)]. Other TAG with different chain length sn-3 fatty acids were synthesized from 1,2-DAG using the appropriate acyl-chloride (Nu-Check Prep, Waterville, MN) [10 (link)]. All TAG were purified from reaction mixtures using preparative TLC after which they were quantified using GC and their purity confirmed using ESI-MS.
Bansal S, & Durrett T.P. (2016). Rapid quantification of low-viscosity acetyl-triacylglycerols using electrospray ionization mass spectrometry. Lipids, 51(9), 1093-1102.
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9

Phospholipase C Detection Assay with SWNT

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Example 4

A study was conducted to test for the presence of phospholipase C (Sigma P7633). In this study, 4 ml of a solution including (6,5)-chiral SWNT at a concentration of 0.5 mg/ml were ultrasonicated alone with phosphatidylcholine at a concentration of 2.5 mg/ml to form a phosphatidylcholine wrapped SWNT.

The phosphatidylcholine wrapped SWNT was diluted to concentrations of 2.5%, 5%, 10%, 15%, and 20% with deionized water and 20 μL of phospholipase C was added. The fluorescence signal from the solution was then measured with respect to time. FIG. 6 shows the response of phosphatidylcholine-functionalized SWNT to different dilutions of 100 mg/mL phospholipase C solution.

US10837045B2. Optical nanosensors for hydrolytic enzyme characterization (2020-11-17). Iowa State University Research Foundation, Inc. [US]. Inventors: Nigel Forest Reuel [US], Nathaniel Kallmyer [US].
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10

Quantifying Complement Regulation by DAF

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5×106 splenocytes from NP-KLH mice were incubated in HL-serum free media for 1h at 37°C, 5% CO2 in the presence or absence of 5ug/ml Phospholipase C (Sigma-Aldrich), washed with PBS 1%FCS and surface DAF expression quantified by flow cytometry. Complement regulatory capacity of DAF-TM was assessed in vitro by incubating 5×106 splenocytes from immunized animals in 20–40% WT or C3−/− mouse serum in Annexin V buffer with 1mM MgCl2 for 20min at 37°C, 5% CO2 and quantifying C3b deposition by flow cytometry.
Cumpelik A., Heja D., Hu Y., Varano G., Ordikhani F., Roberto M.P., He Z., Homann D., Lira S.A., Dominguez-Sola D, & Heeger P.S. (2021). Dynamic regulation of B cell Complement Signaling is Integral to Germinal Center Responses. Nature immunology, 22(6), 757-768.
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