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Phosphatase inhibitor cocktail p0044

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Phosphatase inhibitor cocktail P0044 is a solution designed to inhibit the activity of phosphatases, which are enzymes that remove phosphate groups from proteins. This product is commonly used in various biochemical and cell biology applications to prevent the dephosphorylation of proteins during sample preparation and analysis.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (1 mentions) Hrp linked anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions) Protein assay, by Bio-Rad (1 mentions) Trehalose assay kit k treh 11 12, by Megazyme (1 mentions) Edta free protease inhibitor cocktail, by Roche (1 mentions)

3 protocols using phosphatase inhibitor cocktail p0044

1

Enzymatic Determination of Glucose

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Residual glucose in the supernatants of the chemostat cultures was determined enzymatically using the hexokinase and glucokinase method as previously described57 .
Intracellular and extracellular glucose concentrations in non-chemostat cultures were measured using the Glucose GOD-PAP Liquid Stable Mono-reagent kit (LaborLab Laboratories Ltd. Guarulhos, São Paulo, Brazil), according to the manufacturer’s instructions. For the determination of intracellular glucose concentrations, whole cell extracts were generated by re-suspending mycelial powder in 1 mL of extraction buffer [50 mM Tris base pH 7.0, 50 mM NaF, 1 mM NaVO3, 1 mM DTT, phosphatase inhibitor cocktail P0044 (Sigma) and an EDTA-free protease inhibitor cocktail (Roche)], followed by centrifugation and removal of the glucose-containing supernatants.
dos Reis T.F., Nitsche B.M., de Lima P.B., de Assis L.J., Mellado L., Harris S.D., Meyer V., dos Santos R.A., Riaño-Pachón D.M., Ries L.N, & Goldman G.H. (2017). The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans. Scientific Reports, 7, 45073.
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2

Western Blot Analysis of GFP Protein

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Mycelia were grown in the specified conditions and ground to a fine powder under liquid N2 before being mixed with extraction buffer [50 mM Tris–HCl pH 7.0, 50 mM NaF, 1 mM NaVO3, 1 mM DTT, phosphatase inhibitor cocktail P0044 (Sigma) and the complete mini EDTA-free protease inhibitor cocktail (Roche)]. Samples were centrifuged for 5 min at 14000× g and the protein concentration in the supernatant was determined as described above. 50 μg of total protein were run on pre-made gels, before being transferred to a membrane as described previously10 (link). Membrane blocking and washes, primary and secondary antibody incubation as well as membrane signal detection were carried out as described previously10 (link). The primary rabbit polyclonal IgG antibody anti-GFP (Abcam #ab290) was used in a 1:10000 dilution whereas a 1:5000 dilution was used for the secondary anti-rabbit IgG HRP linked antibody (Cell Signaling Technology, Beverly).
dos Reis T.F., Nitsche B.M., de Lima P.B., de Assis L.J., Mellado L., Harris S.D., Meyer V., dos Santos R.A., Riaño-Pachón D.M., Ries L.N, & Goldman G.H. (2017). The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans. Scientific Reports, 7, 45073.
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3

Quantification of Glycerol and Trehalose in A. fumigatus

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A. fumigatus conidia (1.0 × 105 to 1.0 × 106) were inoculated into liquid YPD (1% yeast extract, 1% polypeptone and 1% glucose) and cultured for 16 h prior to addition of 1/2 vol. 3 M sorbitol (Final concentration: 1 M) and incubated at 37°C for different periods of time. Mycelia were ground in liquid nitrogen and immediately resuspended by inversion in extraction buffer [50 mM: Tris base pH 7.0, 50 mM NaF, 1 mM Na3VO4, 1 mM DTT, phosphatase inhibitor cocktail P0044 (Sigma) and an EDTA-free protease inhibitor cocktail (Roche)] prior to centrifugation for 5 min at 14,000g. The protein concentration of the extracts was measured using the Bio-Rad protein assay according to manufacturer’s instructions. The glycerol and trehalose content within the extracted cell lysate (equivalent to 5, 10 and 20 µg of total protein for the respective assays) was measured using the Free Glycerol Detection ab65337 kit (AbCam) according to the manufacturer’s instructions. The trehalose content was measured using Trehalose Assay kit K-TREH 11/12 (Megazyme) according to the manufacturer’s instructions with an additional standard curve ranging from 0 to 4 µg of trehalose dihydrate.
Alves de Castro P., dos Reis T.F., Dolan S.K., Manfiolli A.O., Brown N.A., Jones G.W., Doyle S., Riaño-Pachón D.M., Squina F.M., Caldana C., Singh A., Del Poeta M., Hagiwara D., Silva-Rocha R, & Goldman G.H. (2016). The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence. Molecular microbiology, 102(4), 642-671.
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