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Phosphatase inhibitor cocktail

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Phosphatase inhibitor cocktail is a laboratory product that contains a mixture of phosphatase inhibitors. The primary function of this cocktail is to inhibit the activity of various phosphatase enzymes in biological samples, thereby preserving the phosphorylation state of proteins during sample preparation and analysis.

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Lab products found in correlation

Protease inhibitor cocktail, by Cell Signaling Technology (15 mentions) Ripa buffer, by Cell Signaling Technology (9 mentions) Protease inhibitor cocktail, by Roche (8 mentions) Protease inhibitor cocktail, by Merck Group (7 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions) β actin, by Merck Group (4 mentions) Revert total protein stain, by LI COR (4 mentions) Image studio, by LI COR (4 mentions) Odyssey clx imager, by LI COR (4 mentions) Benzonase, by Merck Group (4 mentions) Pvdf membrane, by Merck Group (4 mentions) Protease inhibitor, by Roche (3 mentions) Odyssey blocking buffer tbs, by LI COR (3 mentions) Ripa buffer, by Beyotime (3 mentions) Ripa lysis buffer, by Beyotime (3 mentions) Pierce bca protein assay, by Thermo Fisher Scientific (3 mentions) β actin, by Cell Signaling Technology (3 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (3 mentions) Ripa buffer, by Thermo Fisher Scientific (3 mentions) Protease inhibitor, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

Protease/phosphatase inhibitor cocktail (304 citations) Protease and phosphatase inhibitor cocktail (63 citations) Phosphatase inhibitor (44 citations) Protease and phosphatase inhibitors cocktail (4 citations) Protease/Phosphatase Inhibitor Cocktail (100X), (4 citations) Protease and phosphatase inhibitor cocktails I and II (3 citations) Complete protease/phosphatase inhibitor cocktail (3 citations) Protease/Phosphatase Inhibitor Cocktail (1X, (3 citations) Proteinase and phosphatase inhibitor cocktail (3 citations) Protease/phosphatase inhibitors cocktail (2 citations)

84 protocols using phosphatase inhibitor cocktail

1

Quantifying Protein Expression via Western Blot

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Western blot was performed to detect iNOS in the cell fraction as described previously (Zaheer and Lim, 1996 (link)). After exposure to MPP+, cells were lysed in RIPA cell lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, 0.5 % deoxycholate) containing protease (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor cocktails (Cell Signaling Technology, Danvers, MA). Samples containing equal amounts of protein (35 μg) were separated in 10 % SDS-polyacrylamide gels. Proteins were transferred onto PVDF membrane and incubated overnight at 4 °C with primary rabbit polyclonal antibody against NOS2 (Santa Cruz, CA; 1:200 dilutions) followed by secondary antibody conjugated to horseradish peroxidase (HRP). Proteins recognized by the antibody were visualized by enhanced chemiluminescence Femto kit (Thermo Scientific, Rockford, IL). Blots were stripped and reprobed for β-actin (Sigma) as a loading control. Bands intensity was measured by densitometry and quantified using NIH-Image J software.
Khan M.M., Kempuraj D., Zaheer S, & Zaheer A. (2014). Glia Maturation Factor Deficiency Suppresses 1-Methyl-4-Phenylpyridinium-Induced Oxidative Stress in Astrocytes. Journal of molecular neuroscience : MN, 53(4), 590-599.
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2

Cellular Antioxidant Enzyme Assays

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SH-SY5Y cells (1 x 10 5cells/ml) were seeded in a 6well plate followed by incubation with GMF (100 ng/ml) and Aβ (1-42) 10 μM for 24 h. After treatment, cells were washed three times in DPBS and centrifuged at 2,000 g for 10 min at 4 °C. Cells were lysed at 4 °C with radio-immunoprecipitation assay (RIPA) cell lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor cocktails (Cell Signaling Technology, Danvers, MA). Cell lysates were used for analysis of cellular glutathione peroxidase (GPX) (kit no. 703102) and superoxide dismutase (SOD) (kit no. 706002) according to the manufacturer’s instructions (Cayman Chemical Ann Arbor, MI). The activity of GPx was calculated as micromoles of glutathione (GSH) formed per milligram protein, using a molar extinction coefficient of 13.6 × 103/M/cm. The SOD results were expressed as units per milligram protein.
Ahmed M.E., Selvakumar G.P., Kempuraj D., Thangavel R., Mentor S., Dubova I., Raikwar S.P., Zaheer S., Iyer S, & Zaheer A. (2019). Synergy in disruption of mitochondrial dynamics by Aβ (1-42) and Glia maturation factor (GMF) in SH-SY5Y cells is mediated through alterations in fission and fusion proteins. Molecular neurobiology, 56(10), 6964-6975.
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3

Mitochondrial Dysfunction in SH-SY5Y Cells

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SH-SY5Y cells were seeded in T25 cm2 cell culture flasks and incubated with GMF and Aβ peptide for 24 h, thereafter cells were washed with DPBS and adherent cells were detached from the culture flask, cells were collected and centrifuged at 2000 g for 10 min at 4 °C. Cells were lysed at 4 °C with RIPA cell lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor cocktails (Cell Signaling Technology, Danvers, MA). The cell lysate were used for western blotting analysis, Mitochondrial and cytosolic fractions were separated by using the Abcam (Cat no; ab 110170, Cambridge, MA) mitochondria isolation kit according to the manufacturer’s instruction and protein concentration was estimated by the BCA protein assay kit.
Ahmed M.E., Selvakumar G.P., Kempuraj D., Thangavel R., Mentor S., Dubova I., Raikwar S.P., Zaheer S., Iyer S, & Zaheer A. (2019). Synergy in disruption of mitochondrial dynamics by Aβ (1-42) and Glia maturation factor (GMF) in SH-SY5Y cells is mediated through alterations in fission and fusion proteins. Molecular neurobiology, 56(10), 6964-6975.
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4

Immunoblotting Protocol for Protein Analysis

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Immunoblotting procedures were performed as described preciously [62 (link),63 (link)]. Briefly, at the indicated times post infection (p.i.), cells were washed twice with PBS. For IB, cells were lysed by the addition of 1.5 x Laemmli sample buffer containing protease inhibitor (Roche, Product # 04693116001) and phosphatase inhibitor cocktails (Cell Signaling Technology, Cat# 5870). Cells were fully lysed by pipetting followed by sonication. Samples were heated at 95°C and equal amounts of proteins were analyzed on denaturing SDS-polyacrylamide gels. The proteins were transferred to PVDF Immobilon-P membrane (Millipore, Cat# IPVH00010) and probed with specific primary antibody followed by secondary antibody conjugated to horseradish peroxidase. Bands were visualized by chemiluminescence-based detection system (LI-COR Biosciences).
Majumdar T., Chattopadhyay S., Ozhegov E., Dhar J., Goswami R., Sen G.C, & Barik S. (2015). Induction of Interferon-Stimulated Genes by IRF3 Promotes Replication of Toxoplasma gondii. PLoS Pathogens, 11(3), e1004779.
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5

Analyzing STAT4 Signaling in Neutrophils

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BM neutrophils from WT, STAT4fl/flLysMcre, and STAT4fl/flS100A8cre mice were isolated via negative selection kit (STEMCELL Technologies, 19762A) and stimulated with 40 ng/mL IL-12 (Peprotech, 210-12) in RPMI with 10% FBS for the indicated time points. Following stimulation, cells were washed and lysed with lysis buffer (Thermo Fisher Scientific, 78501) contacting protease inhibitor (Thermo Fisher Scientific, 78429) and phosphatase inhibitor cocktails (Cell Signaling Technology, 5870S). Protein content was determined by BCA method (Thermo Fisher Scientific), and 20 μg of protein lysate was resolved on a 4%–20% polyacrylamide gradient gel, followed by transfer to PVDF membrane via semidry transfer. Membranes were blocked at room temperature for 1 hour with TBS-based blocking buffer (LI-COR Biosciences 927-50000). The following primary Abs were incubated at 4°C overnight: STAT4 (Cell Signaling Technology C46B10), p-STAT4 (BD Biosciences 612739), JAK2 (Cell Signaling Technology 3230), p-JAK2 (Cell Signaling Technology, 3771), and GAPDH (Santa Cruz Biotechnology, 32233). Secondary Abs (926-68072 and 926-32211, Li-COR Biosciences) were incubated at room temperature for 1 hour. Images were acquired using the LI-COR Odyssey system. Band intensity was determined using Image Studio Lite software (LI-COR Biosciences).
Mehrpouya-Bahrami P., Moriarty A.K., De Melo P., Keeter W.C., Alakhras N.S., Nelson A.S., Hoover M., Barrios M.S., Nadler J.L., Serezani C.H., Kaplan M.H, & Galkina E.V. (2021). STAT4 is expressed in neutrophils and promotes antimicrobial immunity. JCI Insight, 6(14), e141326.
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6

Tumor Tissue RAD001 Quantification

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The treated mice were sacrificed at 24 h to collect tumor tissues, which were lysed in ice-cold radioimmunoprecipitation buffer supplemented with phosphatase inhibitor cocktails (Cell Signaling, Danvers, MA), centrifuged. The protein was then collected. Protein content and RAD001 concentration were detected by bicinchoninic acid protein assay and HPLC, respectively, which determined the RAD001 concentration normalized by the total protein content.
Yuan Y., Jin P., Wang Y., Zhao X., Hu Q., Wu W., Huang J, & Zhang N. (2020). A dendritic, redox-responsive, supramolecular (Dr.S) system for lysis-triggered delivery for drug-resistant renal cancer. RSC Advances, 10(62), 37826-37833.
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7

Extraction and Preparation of Biological Samples

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From SigmaAldrich (St. Louis, MO, USA), we obtained the following reagents: ethylene-diamine-tetraacetic acid (EDTA), mannitol, sucrose, MOPS, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), and glycerol. From Duchefa Biochemie (Haarlem, The Netherlands), we purchased N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), sodium dodecyl sulfate (SDS), NaCl, and DL-dithiothreitol (DTT). Fetal bovine serum (FBS) was purchased from Corning (Castle Rock, CO, USA). Bovine serum albumin (BSA) was purchased from US Biological Life Sciences (Salem, MA, USA). The proteinase inhibitor cocktail was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and the phosphatase inhibitor cocktail from Cell Signaling Technology (Danvers, MA, USA). Phosphate-buffered saline (PBS) was obtained from Elpis Biotech (Daejeon, Republic of Korea). We used skim milk (BD Difco, BD, Franklin Lakes, NJ, USA). Korea Ginseng Corporation provided the KRGE powder. The stock solutions were made using filtered distilled water. The aliquoted stock solutions (250 mg/mL) were kept at −25 °C in the dark.
Kim H., Moon S., Lee D., Park J., Kim C.H., Kim Y.M, & Choi Y.K. (2023). Korean Red Ginseng-Induced SIRT3 Promotes the Tom22–HIF-1α Circuit in Normoxic Astrocytes. Cells, 12(11), 1512.
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8

Western Blot Analysis of ATO Treatment

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Cell were treated with or without ATO for the indicated time and subsequently lysed in the RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Amresco, Cleveland, Ohio) and/or phosphatase inhibitor cocktail (Cell Signaling Technology). The whole cell lysis was subjected to SDS-PAGE, transferred onto a PVDF membrane, and then immunoblotted with respective primary antibodies. The bound primary antibody were visualized with specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) using an enhanced chemiluminescence (ECL) reagents (Thermo Scientific, Hudson, NH). The bar graphs corresponding to the Western blots were generated through densitometric analysis with Image J software.
Pan C., Zhu D., Zhuo J., Li L., Wang D., Zhang C.Y., Liu Y, & Zen K. (2016). Role of Signal Regulatory Protein α in Arsenic Trioxide-induced Promyelocytic Leukemia Cell Apoptosis. Scientific Reports, 6, 23710.
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9

LYTAC-mediated protein analysis in cells

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Adherent cells were plated (100,000 cells/well in a 24-well plate) one day before the experiment. Cells were incubated with 250 μl of complete growth media with 10 nM LYTAC or controls for indicated time. Cells were then washed with DPBS 3 times and lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche), 0.1% Benzonase (Millipore-Sigma), and phosphatase inhibitor cocktail (Cell Signaling Technologies) on ice for 30 minutes. The cells were scraped, transferred to Eppendorf tubes, and spun down at 21,000g for 15 minutes at 4 °C. The supernatant was collected and the protein concentration was determined by BCA assay (Pierce). Equal amounts of lysates were loaded onto 4–12% Bis-Tris gel and separated by sodium dodecyl sulfate-polacrylamide gel electrophoresis (SDS-PAGE). Then, the gel was transferred onto a nitrocellulose membrane and stained with REVERT Total Protein Stain (LI-COR), then blocked with Odyssey Blocking Buffer (TBS) (LI-COR) for 1 hour at rt. The membrane was incubated with primary antibody overnight at 4 °C, washed 3 times with TBS-T. Subsequently, the membrane was incubated with secondary antibody for 1 hour at rt, and washed 3 times with TBS-T for visualization with an Odyssey CLx Imager (LI-COR). Image Studio (LI-COR) was used to quantify band intensities.
Ahn G., Banik S.M., Miller C.L., Riley N.M., Cochran J.R, & Bertozzi C.R. (2021). LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nature chemical biology, 17(9), 937-946.
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10

Investigating Sigmar1-IRE1α Interaction

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HEK293 cells were transfected with human WT IRE1α (a gift from Fumihiko Urano, Addgene plasmid #20744) (Lipson et al., 2008 (link)), Sigmar1-myc-FLAG transcript variant 1 (#RC201206, Origene), or Sigmar1-myc-FLAG transcript variant 2 (#RC214331, Origene) using Lipofectamine 2000 according to the manufacturer’s protocol. Four days post-transfection, cells were stimulated with 20 μM Linuron. 16 hours later, cells were lysed in ice cold non-denaturing lysis buffer (20 mM Tris-HCl pH=8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA, recipe obtained from Abcam) supplemented with protease inhibitor cocktail (#5871, Cell Signaling, 1:100 working concentration) and phosphatase inhibitor cocktail (#5870, Cell Signaling, 1:100 working concentration). Immunoprecipitation for c-myc was performed for 4 hours at 4C using the following kit (#23620, Thermo Fisher Scientific). SDS-PAGE was then performed as described above. The following antibodies were used: Mouse anti-FLAG M2 (#F1804–200UG, Sigma-Aldrich, 1:1000) and Rabbit anti-IRE1α (14C10) (#3294S, Cell Signaling, 1:1000).
Wheeler M.A., Jaronen M., Covacu R., Zandee S.E., Scalisi G., Rothhammer V., Tjon E.C., Chao C.C., Kenison J.E., Blain M., Rao V.T., Hewson P., Barroso A., Gutiérrez-Vázquez C., Prat A., Antel J.P., Hauser R, & Quintana F.J. (2019). Environmental control of astrocyte pathogenic activities in CNS inflammation. Cell, 176(3), 581-596.e18.
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