Phosphatase inhibitor cocktail 1

Manufactured by Merck Group

Sourced in United States, Germany, Switzerland, Italy

Phosphatase inhibitor cocktail 1 is a solution designed to inhibit the activity of various phosphatases. It contains a combination of chemical compounds that effectively block the enzymatic function of phosphatases in biological samples.

Automatically generated - may contain errors

Lab products found in correlation

Phosphatase inhibitor cocktail 2, by Merck Group (17 mentions) Protease inhibitor cocktail, by Merck Group (16 mentions) Protease inhibitor cocktail, by Roche (9 mentions) Protease inhibitor, by Roche (8 mentions) Complete protease inhibitor cocktail, by Roche (6 mentions) Bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Protease inhibitor, by Merck Group (5 mentions) Mini protease inhibitor cocktail tablet, by Roche (5 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Dc protein assay kit, by Bio-Rad (3 mentions) Nitrocellulose membrane, by Bio-Rad (3 mentions) Cell lysis buffer, by Cell Signaling Technology (3 mentions) Wbkls0100, by Merck Group (3 mentions) Ecl plus, by GE Healthcare (3 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions) Lysis buffer, by Beyotime (3 mentions) Complete protease inhibitor, by Roche (3 mentions) Z ietd fmk, by R&D Systems (2 mentions) Anti caspase 8 551242, by BD (2 mentions)

Close spelling variants of this product

Phosphatase inhibitor cocktail (1110 citations) Phosphatase inhibitors (852 citations) Phosphatase inhibitor cocktails I and II (90 citations) Phosphatases inhibitors (7 citations) Phosphatases (7 citations) Cocktail inhibitor (6 citations) Phosphatase inhibitor cocktails 1 and 3 (3 citations) Phosphatase I inhibitor (2 citations) Phosphatase inhibitor cocktails 1 or 3 (2 citations) Protease inhibitor and phosphatase inhibitor cocktails I and II (2 citations)

90 protocols using phosphatase inhibitor cocktail 1

GFP and Endogenous Protein Immunoprecipitation

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For GFP immunoprecipitation, U2OS cells expressing GFP or GFP-CtIP were harvested in lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, 1 × protease inhibitors [Roche] and 1 × phosphatase inhibitor cocktail 1 [Sigma]). Protein extract (1 mg) was mixed with 50 μl of washed magnetic anti-GFP beads (GFP-Trap_M, Chromotek) and incubated overnight at 4 °C with gentle rocking. Beads were then washed 3 × with lysis buffer, and the precipitate was eluted in SDS sample buffer by boiling the beads and loaded onto a gel.
To immunoprecipitate endogenous proteins, U2OS were scrapped in lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 × protease inhibitors [Roche] and 1 × phosphatase inhibitor cocktail 1 [Sigma]). To degrade DNA, 100 U ml⁻¹ Benzonase (Merck Millipore) was added to protein extracts and incubated for 30 min on ice. Protein extracts (1 mg) were then precleared with magnetic protein A Dynabeads (Novex) under gentle agitation at 4 °C for 30 min. Precleared samples were then incubated with anti-CBX4 antibody or a rabbit IgG (Sigma) as a control for 1.5 h at 4 °C and Dynabeads were added afterwards and were incubated for 1 h at 4 °C under gentle agitation. Beads were then washed three times with lysis buffer, and the precipitate was eluted in Laemmli buffer.

Soria-Bretones I., Cepeda-García C., Checa-Rodriguez C., Heyer V., Reina-San-Martin B., Soutoglou E, & Huertas P. (2017). DNA end resection requires constitutive sumoylation of CtIP by CBX4. Nature Communications, 8, 113.

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Liver Protein Extraction and Western Blot

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Ground livers were lysed in buffer containing 10mM Tris-HCL pH 8.0, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium de-oxycholate, 0.1% SDS, 140mM NaCl, protease inhibitor cocktail (cOmplete, Roche), and phosphatase inhibitor (Phosphatase Inhibitor Cocktail 1, Sigma), sonicated, normalized using a Bradford Assay (Bio-Rad), and resolved by SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes using semi-dry transfer cells (Bio-Rad), and blocked in 5% milk.
Cell cultures were washed 1x with PBS, lysed in buffer containing 2% Triton x-100, 100mM Tris-HCl pH 7.4, 300mM NaCl, 10% Glycerol, 5mM EDTA, 5mM EGTA, 2mM DTT, protease and phosphatase inhibitors (cOmplete, Roche; Phosphatase Inhibitor Cocktail 1, Sigma), spun at 21000 g for 10 min at 4°C, mixed with SDS-PAGE loading buffer and resolved by electrophoresis.

Gassaway B.M., Cardone R.L., Padyana A.K., Petersen M.C., Judd E.T., Hayes S., Tong S., Barber K.W., Apostolidi M., Abulizi A., Sheetz J.B., K.s.h.i.t.i.z., Aerni H.R., Gross S., Kung C., Samuel V.T., Shulman G.I., Kibbey R.G, & Rinehart J. (2019). Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production. Cell reports, 29(11), 3394-3404.e9.

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Kinase Activity Assay for CDK1/cyclin B1 and Aurora B/INCENP

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Recombinant CDK1/cyclin B1 and Aurora B/INCENP were purchased from Carna Biosciences (Kobe, Japan), and recombinant histone H1 and kemptide were purchased from Merck Millipore (Billerica, MA, USA). The kinase and substrate were incubated in kinase buffer (50 mM Tris, pH 7.5, 10 mM NaCl, 10 mM MgCl₂, 100 μg ml⁻¹ BSA, and 1 mM DTT) containing phosphatase inhibitor cocktail 1 (Sigma) and 50 mM ATP (Sigma) for 1 h at 30 °C. The level of unincorporated ATP was measured with Kinase-Glo Luminescent Kinase assays (Promega, Madison, WI, USA) using an Infinite M200 (Tecan, Männedorf, Switzerland). Synthetic peptides were produced at Peptron (Daejeon, Korea). The synthetic peptides used were as follows: T603: LKELKFLTPVRRSRR, T603A: LKELKFLAPVRRSRR, S608: FLTPVRRSRRIQDKT, S608A: FLTPVRRARRIQDKT.

Yoo B.H., Kang D.S., Park C.H., Kang K, & Bae C.D. (2017). CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity. Experimental & Molecular Medicine, 49(7), e354-.

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Apoptosis Pathway Antibodies and Regulators

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Anti-caspase 8 (551242), and anti-RIP1 (551042) antibodies were from BD
Biosciences. Anti-caspase 8 (52183, for immunofluorescence), anti-RIPK3 (56164),
and anti-adenine nucleotide translocase (ANT, 109864) antibodies were from
Abcam. Anti-CypD (AP1035) antibody was from Calbiochem. Anti-hexokinase II
(2106), anti-BAX (2772), anti-Bcl-x_L (2762), anti-caspase 3 (9662),
anti-caspase 9 (9502), anti-BID (2002), anti-GAPDH (2118), and anti-caspase 8
(Asp387, 14071, for flowcytometry) were from Cell Signaling Technology.
Anti-VDAC (SP5361P) antibody was from Acris Antibodies. Active caspase 3 for
flowcytometry was from BD. Anti-human mitochondria antibody (HMS-0100) was from
Immunovision. Dylight 650-conjugated Donkey anti-rabbit IgG antibody and Alexa
fluro 488-conjugated Donkey anti-human IgG antibody were from Jackson Immuno
Research Laboratory. The selective caspase 8 inhibitor z-IETD-FMK (FMK007) was
from R&D Systems. Gammabind protein G sepharose (17-0885-01) was from GE
Health Care. Cyclosporin A (30024), Tiron (89460) and Protease Inhibitor
Cocktail (P2714) from Calbiochem and Phosphatase Inhibitor Cocktail 1 (P2850)
was from SIGMA-ALDRICH. Mitochondria/Cytosol Fractionation Kit (ThermoFisher
Scientific Mitochondrial Isolation kit #89874) and MitoTracker Red
CMXRos (M7512), MitoTracker Red CM-H₂XRos (M7513), and LIVE/DEAD
Fixable Dead Cell Stain Kits (MP34955) from Invitrogen.

Zhao X., Khan N., Gan H., Tzelepis F., Nishimura T., Park S.Y., Divangahi M, & Remold H.G. (2017). Bcl-xL mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages. Mucosal immunology, 10(6), 1553-1568.

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Whole Cell Extract Preparation and Immunoblotting

Cited 2 times

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Preparation of whole cell extracts and immunoblotting were carried out as described previously (12 (link), 27 (link), 63 (link)). Briefly, cells were harvested and lysed in a lysis buffer [10 mM tris-HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% Na-deoxycholate supplemented with complete protease inhibitors] (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 1 from Sigma-Aldrich). After thorough mixing and incubation at 4°C for 2 hours, lysates were then centrifuged at 12,000g at 4°C for 20 min. Supernatants were collected and stored in aliquots at −80°C. Immunoblots were carried out following standard procedures, and immunoreactivity was detected using enhanced chemiluminescence reaction (170-5061, Bio-Rad) under ChemiDoc MP System (Bio-Rad).

Ghosh A., Bhattacharjee S., Chowdhuri S.P., Mallick A., Rehman I., Basu S, & Das B.B. (2019). SCAN1-TDP1 trapping on mitochondrial DNA promotes mitochondrial dysfunction and mitophagy. Science Advances, 5(11), eaax9778.

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GFP-selector Immunoprecipitation Protocol

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Cell pellets were lysed in 300 mM NaCl buffer (20 mM HEPES-KOH pH 7.9, 300 mM NaCl, 3 mM MgCl_2, 0.1% NP-40, 1X protease inhibitor cocktail (Roche), 1X phosphatase inhibitor cocktail-1 (Sigma) and 10 mM sodium butyrate) for 30 min at 4 °C with rotation. After a 15 min centrifugation at 12,000 g at 4 °C, the supernatants were collected and separated into input and immunoprecipitate (IP) lysates. IP lysates were mixed with GFP-selector (pre-washed with 300 mM NaCl buffer three times, NanoTag) and rotated overnight at 4 °C. After washing with 300 mM NaCl buffer, IPs were released by adding Laemmli sample buffer to beads and boiling for 5 min at 100 °C.

Ibrahim Z., Wang T., Destaing O., Salvi N., Hoghoughi N., Chabert C., Rusu A., Gao J., Feletto L., Reynoird N., Schalch T., Zhao Y., Blackledge M., Khochbin S, & Panne D. (2022). Structural insights into p300 regulation and acetylation-dependent genome organisation. Nature Communications, 13, 7759.

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Western Blot Protein Detection Protocol

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Cells were lysed with RIPA (Radioimmunoprecipitation Assay) buffer (50mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) plus protease inhibitor cocktail (Roche, Mannheim Germany) and phosphatase inhibitors (phosphatase inhibitor cocktail 1 from Sigma-Aldrich). Lysates were loaded onto SDS-PAGE and separated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane from Millipore (Billerica, MA, USA) and probed with the primary antibody diluted in 2% milk in PBS (Phosphate-Buffered Saline) followed by HRP-conjugated secondary antibody, as previously described [23 (link)]. Bands were visualized using Western blot Luminol Reagent (Santa Cruz), WesternBright ECL kit (Advansta, Menlo Park, CA, USA), Clarity (BioRad, Hercules, CA, USA), or Clarity Max (BioRad, Hercules, CA, USA), and signals were captured on a film or using Bio-Rad ChemiDoc MP Imaging Systems (Hercules, CA, USA).

Romano R., Calcagnile M., Margiotta A., Franci L., Chiariello M., Alifano P, & Bucci C. (2021). RAB7A Regulates Vimentin Phosphorylation through AKT and PAK. Cancers, 13(9), 2220.

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Drosophila Adult Head Proteomic Analysis

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Drosophila adult head extract was obtained by homogenizing adult heads collected on dry ice in high EDTA RIPA buffer (50 mM Tris-HCl, pH7.5, 1% NP-40, 0.5% NaDoc, 150 mM NaCl, 0.1% SDS, 10 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 250 nM cycloporin A, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 1 (Sigma) using mortar and pestle. 10–20 μg protein homogenate was separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies were diluted in blocking solution as following: rabbit anti-p-Synj, 1:5000; guinea pig anti-Endo(GP60), 1:200; rabbit anti-Dap160, 1:5000; rabbit anti-Mnb, 1:500, rabbit anti-Synj-1-rabbit. 1:200; mouse anti-dynamin, 1:200; mouse anti-complex V, 1:10,000 (MitoSciences); rabbit anti-phosphothreonine, 1:200 and rabbit anti-phosphoserine, 1:200 (EMD Millipore). To detect the amount of protein loading, nitrocellulose membrane was stained with Direct Blue 71. All values were normalized to control done within the same experimental set. Intensity of each band was quantified using Image J. Full images of key western blots are shown in Supplementary Fig. 7.

Chen C.K., Bregere C., Paluch J., Lu J., Dickman D.K, & Chang K.T. (2014). Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase. Nature communications, 5, 4246.

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Affinity Purification of Biotinylated Proteins

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HEK293 cells were transfected with pDNA3.1-UL8-V5 or pDNA3.1-UL8-V5-TbID for 48 hours with Lipofectamine 2000, and then, 50 µM of biotin was added to the media for 6 hours. Cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 8, 1% Nonidet P-40, 150 mM NaCl, and 1× protease inhibitor cocktail (P8340, Sigma), 1× phosphatase inhibitor cocktail 1 (P2850; Sigma), 1× phosphatase inhibitor cocktail 2 (P5726; Sigma), and insoluble debris were pelleted at 10,000 × g for 10 min at 4°C. One milligram of protein lysates was incubated at 4°C overnight with 500 µL of streptavidin conjugated to magnetic beads (New England BioLabs, Ipswich, MA, USA). Beads were washed once in 1.5 mL of wash buffer 1 (2% SDS in H₂O), once with wash buffer 2 [0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM HEPES (pH 7.5)], once with wash buffer 3 [250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8.1)], and then twice with wash buffer 4 [50 mM Tris (pH 7.4), 50 mM NaCl]. To evaluate sample integrity, 10% of the total was retained for immunoblots. The remaining beads were spun at 2,000 × g and resuspended in 50 µL of 50 mM ammonium bicarbonate for mass spectrometry.

Dirck A., Diggins N.L., Crawford L.B., Perez W.D., Parkins C.J., Struthers H.H., Turner R., Pham A.H., Mitchell J., Papen C.R., Malouli D., Hancock M.H, & Caposio P. (2023). HCMV UL8 interaction with β-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells. Journal of Virology, 97(10), e01241-23.

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Protein Extraction and Western Blot Analysis

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DRGs were freshly dissected out and subject to homogenization for protein extraction in T-PER buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (P8340, 1:100, Sigma-Aldrich) and phosphatase inhibitor cocktail 1 (P2850, 1:100, Sigma-Aldrich). After centrifugation at 20,200 g for 10 min at 4 °C, the protein concentration in the supernatant was determined using Bio-Rad DC protein assay kit. Protein extracts were then separated on SDS-PAGE gel and transferred to a nitrocellulose membrane for immunoblotting. The primary antibodies used for western blot were rabbit anti-p-Akt (1:1000, Cell Signaling, Cat# 4060), rabbit anti-Akt (1:1000, Millipore Sigma, Cat# SAB4500800), rabbit anti-p-CREB (1:1000, Cell Signaling, Cat# 9198), and mouse anti-beta actin (1:5000, Sigma, Cat# A2228). The secondary antibodies were anti-rabbit IgG, HRP-linked (1:2500, Cell Signaling, Cat# 7074) and anti-mouse IgG, HRP-linked (1:2500, Cell Signaling, Cat# 7076). The proteins of interests were blotted followed by striping and re-blotting process to visualize the internal controls. The bands were identified by enhanced chemiluminescence (ECL) and analyzed by Imaging J.

Madar J., Tiwari N., Smith C., Sharma D., Shen S., Elmahdi A, & Qiao L.Y. (2023). Piezo2 regulates colonic mechanical sensitivity in a sex specific manner in mice. Nature Communications, 14, 2158.

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