Cocktail of phosphatase inhibitors

Manufactured by Roche

Sourced in Switzerland

This product is a cocktail of phosphatase inhibitors. It is designed to inhibit the activity of phosphatase enzymes in biological samples. The specific components and their concentrations are not provided.

Automatically generated - may contain errors

Lab products found in correlation

Cocktail of proteinase inhibitors, by Roche (5 mentions) Nitrocellulose membrane, by Bio-Rad (4 mentions) Ripa buffer, by Qiagen (3 mentions) Proteinase inhibitor, by Roche (2 mentions) Protein extraction reagent, by Thermo Fisher Scientific (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Complete protease inhibitor tablet, by Roche (2 mentions) β actin, by Abcam (1 mentions) β actin, by Bio-Rad (1 mentions) Quantity one v4, by Bio-Rad (1 mentions) Semi dry apparatus, by Bio-Rad (1 mentions) Mmp 13, by Santa Cruz Biotechnology (1 mentions) Anti clu, by Santa Cruz Biotechnology (1 mentions) Eif3i, by Abcam (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Ip lysis buffer, by Thermo Fisher Scientific (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions) Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Gapdh antibody, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Cocktails of protease and phosphatase inhibitors Phosphatase inhibitors cocktail Phosphatase inhibitor cocktail Cocktail inhibitor Inhibitor cocktails Phosphatase inhibitors Phosphatases

11 protocols using cocktail of phosphatase inhibitors

Quantitative Analysis of LBP Protein Expression

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Western blot analysis was performed according to a previous study (29 (link)). Briefly, tissue samples were homogenized in a RIPA buffer (Qiagen, Inc.) supplemented with a cocktail of proteinase inhibitors and with a cocktail of phosphatase inhibitors (both Roche Diagnostics). Protein concentrations were determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (20 µg/lane) were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% bovine serum albumin/PBS at room temperature for 1 h, the membranes were incubated with primary LBP antibody (anti-LBP antibody; 1:1,000 dilution; cat. no. ab169776; Abcam) and β-actin (1:1,000 dilution; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C. After washing with Tween-20 (0.05%) in PBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:2,000 dilution; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. LBP and β-actin were detected using ECL development solution (Pierce; Thermo Fisher Scientific, Inc.). LBP and β-actin expression levels were determined using Quantity One v4.6.2 software (Bio-Rad Laboratories, Inc.). β-actin was used as a loading control.

Cai Q.Y., Jiang J.H., Jin R.M., Jin G.Z, & Jia N.Y. (2019). The clinical significance of lipopolysaccharide binding protein in hepatocellular carcinoma. Oncology Letters, 19(1), 159-166.

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Protein Extraction and Western Blotting

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Protein extraction and western blotting were performed as previously described[14 (link)]. Cell lysates were extracted with the T-PER tissue protein extraction reagent (Pierce, Rockford, IL) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane using a Bio-Rad SemiDry apparatus. After blocking for nonspecific binding, the membranes were incubated with anti-CLU (1:200 dilution; Santa Cruz Biotechnology), MMP13 (1:200 dilution; Santa Cruz Biotechnology), p-Akt (1:1000 dilution; Cell Signaling Technology), Akt (1:1000 dilution; Cell Signaling Technology), EIF3I (1:800 dilution; Abcam, Hong Kong, China) or GAPDH (1:5000 dilution; Kang-Chen, Shanghai, China) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, protein bands were visualized using chemiluminescence detection.

Wang C., Jin G., Jin H., Wang N., Luo Q., Zhang Y., Gao D., Jiang K., Gu D., Shen Q., Huo X., Hu F., Ge T., Zhao F., Chu W., Shu H., Yao M., Cong W, & Qin W. (2014). Clusterin facilitates metastasis by EIF3I/Akt/MMP13 signaling in hepatocellular carcinoma. Oncotarget, 6(5), 2903-2916.

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Western Blot Analysis of NPC2 and NPC1L1

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Western blot was performed according to previous study 15 . Briefly, tissue samples were homogenated in a RIPA buffer (Qiagen, China) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). The protein concentrations were determined using the Bicinchoninic Acid (BCA) Kit (Pierce). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were incubated with primary NPC2 antibodies (A5413; Abclonal, USA) at 1:1500, or NPC1L1-HRP (ab201773; Abcam, USA): 1:2000) overnight at 4 °C. After washing the membrane, NPC1L1 was visualised using ECL development solution. For detection of NPC2, secondary antibodies incubated at room temperature for 1~2 h and NPC2 was visualised using ECL development solution.

Chen K.J., Jin R.M., Shi C.C., Ge R.L., Hu L., Zou Q.F., Cai Q.Y., Jin G.Z, & Wang K. (2018). The prognostic value of Niemann-Pick C1-like protein 1 and Niemann-Pick disease type C2 in hepatocellular carcinoma. Journal of Cancer, 9(3), 556-563.

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Immunoprecipitation for Cellular Protein Analysis

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Immunoprecipitation (IP) was performed using the A549, H1703, and HEK 293 T cells. The protein lysates were extracted with IP lysis buffer (Thermo) containing a cocktail of phosphatase inhibitors (Roche, Basel, Switzerland) and PMSF (Sigma) for 30 min on ice, followed by centrifugation at 12,000 × g for 15 min. The proteins in the lysates were incubated with protein A/G magnetic beads (Thermo) and the indicated primary antibody or normal IgG overnight at 4 °C. The beads were washed three times in washing buffer. Loading buffer (2 ×) was mixed with the beads and boiled for 10 min. Then, the samples were used for western blot analysis.

Sun Y., Gao Y., Dong M., Li J., Li X., He N., Song H., Zhang M., Ji K., Wang J., Gu Y., Wang Y., Du L., Liu Y., Wang Q., Zhai H., Sun D., Liu Q, & Xu C. (2023). Kremen2 drives the progression of non-small cell lung cancer by preventing SOCS3-mediated degradation of EGFR. Journal of Experimental & Clinical Cancer Research : CR, 42, 140.

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Western Blot Analysis of Melanocyte Proteins

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The MCs were seeded in the 60 mm cell culture dish and allowed to grow to 60%-80%, and were stimulated by α-MSH and/or siRNA for 48 h. Then, the total protein of cells was extracted using RIPA lysis buffer with a cocktail of proteinase inhibitors (Roche, Mannheim, Germany) and a cocktail of phosphatase inhibitors (Roche, Mannheim, Germany) according to its protocol. 30 μg total protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. After blocking by 0.1% BSA, the membranes were incubated with primary antibody TYR (#ab180753, Abcam, Cambridge, UK) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, AP0066, Bioworld Technology, Inc., Minnesota, USA) overnight at 4° C and followed by an incubation period of 1 h at room temperature with secondary antibody (1:10000, LI-COR Biosciences, USA). Bands were detected by the enhanced LI-COR Odyssey infrared imaging system (LI-COR Biosciences, NE, USA).

Jiang L., Huang J., Hu Y., Lei L., Ouyang Y., Long Y., Li H., Li S., Yang L., Yang Y., Huang L., Xiang H., Xiao R., Chen J, & Zeng Q. (2020). Identification of the ceRNA networks in α-MSH-induced melanogenesis of melanocytes. Aging (Albany NY), 13(2), 2700-2726.

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Western Blot Analysis of Protein Expression

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Briefly, total protein of tissue samples and cell lines was extracted using protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science) according to its protocol. Equal amount of total protein (20 μg) was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking for nonspecific binding, the membranes were incubated with following primary antibodies: E-cadherin (Abcam, 1:1,000), Vimentin (Abcam, 1:1,000), N-cadherin (Cell Signaling Technology, 1:1,000), FMN2 (Abcam, 1:1,000), TSG101 (1:1,000, Proteintech, Chicago, USA), HSP70 (1:2,000, Proteintech, Chicago, IL, USA), and GAPDH antibody (1:2,000; Cell Signaling Technology) overnight at 4°C. Then PVDF membranes were incubated with the appropriate secondary antibody (Santa Cruz Biotechnology, 1:10,000) for 1 h at room temperature. Bands were detected by a Bio-rad ChemiDoc XRS system.

Shan G., Shao B., Liu Q., Zeng Y., Fu C., Chen A, & Chen Q. (2020). circFMN2 Sponges miR-1238 to Promote the Expression of LIM-Homeobox Gene 2 in Prostate Cancer Cells. Molecular Therapy. Nucleic Acids, 21, 133-146.

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CTCF and BORIS Protein Interaction

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Cells were collected in immunoprecipitation lysis buffer (50 mM Tris-HCl buffer (pH 7.4), 100 mM NaCl, 1% Triton-100, 1 mM PMSF), containing a 1× complete protease inhibitor tablet (Roche) per 10 ml buffer and a cocktail of phosphatase inhibitors (Roche). Homogenates were centrifuged at 20,000g for 10 min at 4 °C to obtain supernatants. DNase I (approximately 1 U ml⁻¹) was used to degrade DNA in supernatants by incubation for 1 h at room temperature. Co-immunoprecipitation of endogenously expressed proteins was performed using protein A Dynabeads (Invitrogen), according to the manufacturer’s instructions. In brief, antibody-conjugated Dynabeads were incubated with purified cell lysates to immunoprecipitate the target antigen. Antibodies used for immunoprecipitation were CTCF (3417, Cell Signaling Technology) and BORIS (NBP2–52405, NOVUS Biologicals). The elution step was conducted by heating the beads for 10 min at 95 °C in lithium dodecyl sulfate (LDS) sample buffer with reducing agent (Invitrogen), after which western blotting was performed using the following antibodies: CTCF (3417, Cell Signaling Technology) and BORIS (9851, Active Motif).

Debruyne D.N., Dries R., Sengupta S., Seruggia D., Gao Y., Sharma B., Huang H., Moreau L., McLane M., Day D.S., Marco E., Chen T., Gray N.S., Wong K.K., Orkin S.H., Yuan G.C., Young R.A, & George R.E. (2019). BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells. Nature, 572(7771), 676-680.

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Western Blot Analysis of LUSC Proteins

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Briefly, total protein of LUSC tissue samples and cell lines was extracted using protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science) according to its protocol. Equal amount of total protein (20 μg) was separated by 10% SDS–PAGE and transferred onto a PVDF membrane. After blocking for nonspecific binding, the membranes were incubated with antibody FOXM1 (1:3000 dilution; proteintech; 13147-1-AP), CENPA (1:1000 dilution; Abcam; ab45694), CENPB (1:1000 dilution; Abcam; ab25734), CCNB1 (1:1000 dilution, Cell Signaling Technology; #4134), or β-actin (1:10000 dilution; Sigma; A2228) overnight at 4 °C and followed by an incubation period of 1 h at room temperature with secondary antibody (1:4000, Bioword; 20330016-1). Bands were detected by a Bio-rad ChemiDoc XRS system. Full scans of the western blots shown in Figs. 4e, f, 5g and 6a, b and Supplementary Figs. 5c and 6c are provided in Source Data file.

Cheng Z., Yu C., Cui S., Wang H., Jin H., Wang C., Li B., Qin M., Yang C., He J., Zuo Q., Wang S., Liu J., Ye W., Lv Y., Zhao F., Yao M., Jiang L, & Qin W. (2019). circTP63 functions as a ceRNA to promote lung squamous cell carcinoma progression by upregulating FOXM1. Nature Communications, 10, 3200.

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Western Blot Analysis of Protein Targets

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Cell or tumour tissue was lysed in NP-40 buffer (Invitrogen) containing a 1× complete protease inhibitor tablet (Roche) per 10 ml buffer and a cocktail of phosphatase inhibitors (Roche). Protein concentration was measured using the DC Protein Assay (Bio-Rad); protein (50 μg) was denatured in LDS sample buffer with reducing agent (Invitrogen), separated on precast 4–12% Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were incubated in blocking buffer (5% dry milk in TBS with 0.2% Tween-20) for 1 h, and then incubated in the primary antibody in blocking buffer overnight at 4 °C. Chemiluminescent detection was performed with the appropriate secondary antibodies and developed using Genemate Blue ultra-autoradiography film (VWR). The actin loading controls for the protein samples shown in the immunoblots of the following panels (two independent mouse tumour samples, and cell lines representative of two independent experiments) are the same because the samples were run on a single gel but probed for pALK, ALK (Extended Data Fig. 2a), MYCN (Extended Data Fig. 3e) and BORIS (Extended Data Fig. 4a), respectively.

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Western Blot Analysis of Liver Inflammation Markers

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The protein expression levels of TNF-α, IL-6, NF-κB, p-NF-κB and β-actin in the liver tissues obtained from rats in different groups were detected by western blot analysis. The liver tissues were incubated in lysis buffer supplied with a cocktail of phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Total protein was quantified using the bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The protocol and semiquantitative analysis were performed according the protocol of our previous study (14 ). The following antibodies were used: TNF-α, IL-6, NF-κB, p-NF-κB (rabbit antibody; dilution 1:1,000) and β-actin (rabbit antibody; dilution 1:1,000). The secondary antibodies used were the following: Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; cat. no. 7074; dilution 1:5,000; Santa Cruz Biotechnology, Inc.) or anti-mouse IgG (cat. no. 7076; dilution 1:5,000; Santa Cruz Biotechnology, Inc.).

Chen B., Ma Y., Xue X., Wei J., Hu G, & Lin Y. (2019). Tetramethylpyrazine reduces inflammation in the livers of mice fed a high fat diet. Molecular Medicine Reports, 19(4), 2561-2568.

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