Log In Sign up
The largest database of trusted experimental protocols

Halt phosphatase inhibitor single use cocktail

Manufactured by Thermo Fisher Scientific
Visit Supplier
Sourced in United States

Halt™ Phosphatase Inhibitor Single-Use Cocktail is a ready-to-use formulation designed to inhibit protein phosphatase activity during protein extraction and analysis. It is provided in a convenient single-use format.

Automatically generated - may contain errors

Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (5 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Protease inhibitor cocktail, by Merck Group (3 mentions) Ripa buffer, by Merck Group (3 mentions) Halt protease inhibitor single use cocktail, by Thermo Fisher Scientific (2 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Rabbit anti desmin, by Abcam (1 mentions) Rabbit anti cd31, by Abcam (1 mentions) Complete lysis m kit, by Roche (1 mentions) Dc kit, by Bio-Rad (1 mentions) Rabbit anti calnexin, by Abcam (1 mentions) Rabbit anti ki67, by Abcam (1 mentions) Tissueruptor, by Qiagen (1 mentions) Las 4000 system, by Fujifilm (1 mentions) Ab174830, by Abcam (1 mentions) Ab8226, by Abcam (1 mentions) Pierce ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)

13 protocols using halt phosphatase inhibitor single use cocktail

1

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Pieces of whole liver were homogenized using a tissue homogenizer (TissueRuptor, Qiagen, Germantown, MD), followed by centrifugation. Protein extraction from cells or whole liver was performed using lysis buffer complemented with protease inhibitor (Complete Lysis M kit, Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor (Halt Phosphatase Inhibitor Single-Use Cocktail, Thermo Scientific, Waltham, MA). Protein concentration was measured using a Bio-Rad DC kit (Bio-Rad Laboratories, Hercules, CA) and lysates were then subjected to immunoblot analysis. Blots were developed with the ECL Detection System (Amersham, Pharmacia Biotech, Buckinghamshire, England). Densitometry of bands was performed with ImageJ software (rsbweb.nih.gov/ij, NIH, Bethesda, MD). Western blot membranes were incubated with the following primary antibodies: Rabbit anti-β-PDGFR (1:500) and anti-phospho-β-PDGFR (1:500) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, rabbit anti-CD31 (1:500), rabbit anti-Ki67 (1:2500), rabbit anti-Desmin (1:1000) and rabbit anti-Calnexin (1:3,000) were from Abcam, Cambridge, England). The reactions were detected with the Fujifilm LAS-4000 system (Fujifilm Life Science, Stamford, CT), using a horseradish peroxidase-conjugated secondary antibody (Anti-rabbit IgG, Cell Signaling, Danvers, MA).
Kocabayoglu P., Zhang D.Y., Kojima K., Hoshida Y, & Friedman S.L. (2015). Induction and Contribution of β-PDGFR Signaling by Hepatic Stellate Cells to Liver Regeneration after Partial Hepatectomy in Mice. Liver international : official journal of the International Association for the Study of the Liver, 36(6), 874-882.
+ Open protocol
+ Expand
2

Analyzing Protein Expression in Cell Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols
After removing spent media and washing cells with cold phosphate buffered saline (PBS), the cells were incubated with cold Pierce RIPA lysis buffer (Thermo Scientific, Hudson, NH) containing Halt™ Protease Inhibitor Single-Use Cocktail, Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and 1 mM dithiothreitol (DTT) for 15 min with occasional swirling. Then, the cells were scraped, homogenized with a 26-gauge needle and vortexed at the highest setting for 1 min; the lysates were cleared by centrifuging at 16,000 g at 4°C for 15 min. Protein concentration was determined with the bicinchoninic acid (BCA) method (BCA Protein Assay - Reducing Agent Compatible; Thermo Scientific). Proteins were separated on NuPAGE 4–12% Bis Tris gel electrophoresis (Life Technologies), and transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose; Life Technologies) using iBlot Gel Transfer Device (Life Technologies). The membrane was probed with monoclonal rabbit antibodies, anti-HMGCR (1:500, ab174830, Abcam Inc., Cambridge, MA), anti-vimentin (1:1000, ab92547, Abcam), and anti-E-cadherin (1:1000, 24E10, Cell Signaling Technology, Beverly, MA). A monoclonal mouse antibody to β-actin (1:500, ab8226, Abcam) was used as a loading control. Immunodetection was performed using the iBlot Western Detection chromogenic kit (Life Technologies).
Warita K., Warita T., Beckwitt C.H., Schurdak M.E., Vazquez A., Wells A, & Oltvai Z.N. (2014). Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion. Scientific Reports, 4, 7593.
+ Open protocol
+ Expand
3

Protein Expression Profiling of ESCC Xenograft Tumors

Check if the same lab product or an alternative is used in the 5 most similar protocols
Proteins were extracted from ESCC xenograft tumors and small intestine tissue using Trident RIPA Lysis Buffer (GeneTex, Inc) with the addition of Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific™) and Protease Inhibitor Cocktail (Sigma). Protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific™). Antibodies for PARP (9532), Caspase-8 (9746), Caspase-9 (9502), Caspase-3 (9662), TNFR1 (3736), CYLD (8462), JNK (9252), p-JNK (4668), Apaf-1 (8969), Bax (5023), Bcl-2 (15071), Cytochrome c (4280), NF-κB (8242), and Myd88 (4283) were purchased from Cell Signaling. TLR4 antibody (ab13556) was obtained from Abcam. UltraSignal Hypersensitive ECL Western Blotting Substrate (4A Biotech, 4AW011-1000).
Zhou P., Li Z., Xu D., Wang Y., Bai Q., Feng Y., Su G., Chen P., Wang Y., Liu H., Wang X., Zhang R, & Wang Y. (2019). Cepharanthine Hydrochloride Improves Cisplatin Chemotherapy and Enhances Immunity by Regulating Intestinal Microbes in Mice. Frontiers in Cellular and Infection Microbiology, 9, 225.
+ Open protocol
+ Expand
4

Protein Extraction and Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols
Total protein was extracted using 1x RIPA buffer (Millipore, North Ryde, NSW) with 1 μL/mL protease inhibitor cocktail (Sigma-Aldrich, Castle Hill, NSW) and 10 μL/mL Halt Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific, Rockford, IL). Total protein content was determined using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions.
Wallace M.A., Della Gatta P.A., Ahmad Mir B., Kowalski G.M., Kloehn J., McConville M.J., Russell A.P, & Lamon S. (2016). Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation. Frontiers in Physiology, 7, 7.
+ Open protocol
+ Expand
5

Protein Expression Analysis of Purine Biosynthesis Enzymes

Check if the same lab product or an alternative is used in the 5 most similar protocols
To assess protein expression of the 2×Strep-tagged fusions, 293T cells grown in normal medium were seeded at 4 × 105 cells per well in a 6-well plate. At 70–80% confluency, cells were transfected with 4.0 μg of pCI-neo-Strep-tag II plasmid in a 1:2.5 ratio with Lipofectamine 2000 as according to manufacturer’s protocol. After 48 h of expression, cells were harvested by trypsinization, washed once with 1× Dulbecco’s phosphate buffered saline (DPBS), and lysed in 100 μL of lysis buffer [50 mM sodium phosphate dibasic pH 8.0, 300 mM sodium chloride, 5% (v/v) glycerol, 1% (v/v) Triton X-100, 1× Halt phosphatase inhibitor single-use cocktail (Thermo-Fisher Scientific), and 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)] for 30 min on ice. Soluble lysate (40 μg for PPAT and PFAS detection and 30 μg for remaining proteins) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 8% polyacrylamide gel), transferred to a PVDF membrane, and probed for the presence of PPAT (LifeSpan Biosciences, catalog no.: LSC80815), GART (Bethyl Laboratories, catalog no.: A304-311A), PFAS (Bethyl Laboratories, catalog no.: A304-220A), PAICS (Bethyl Laboratories, catalog no.: A304-546A), ADSL (Bethyl Laboratories, catalog no.: A304-778A), or ATIC (Bethyl Laboratories, catalog no.: A304-271A).
Liu C., Knudsen G.M., Pedley A.M., He J., Johnson J.L., Yaron T.M., Cantley L.C, & Benkovic S.J. (2019). Mapping Post-Translational Modifications of de Novo Purine Biosynthetic Enzymes: Implications for Pathway Regulation. Journal of proteome research, 18(5), 2078-2087.
+ Open protocol
+ Expand
6

Immunoprecipitation of Tyrosine-Phosphorylated AXL

Check if the same lab product or an alternative is used in the 5 most similar protocols
IP was performed using Protein A-Sepherose 4A beads (Life Technologies). Low salt IP-lysis buffer was enriched with Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and Halt Protease Inhibitor Single-Use Cocktail (Thermo Scientific). Immuncomplexes were prepared with 200 μg protein and 1:50 dilution of anti-AXL (C89E7) rabbit antibody (Cell Signaling). Assay was performed according to previous protocol (14). Detection of RTK-AXL phosphorylation was determined using antibody 4G10 mouse anti-tyrosine antibody (Max Planck Institute of Biochemistry, Dilution 1:2.000).
Onken J., Torka R., Korsing S., Radke J., Krementeskaia I., Nieminen M., Bai X., Ullrich A., Heppner F, & Vajkoczy P. (2016). Inhibiting receptor tyrosine kinase AXL with small molecule inhibitor BMS-777607 reduces glioblastoma growth, migration, and invasion in vitro and in vivo. Oncotarget, 7(9), 9876-9889.
+ Open protocol
+ Expand
7

Comprehensive Protein Extraction and Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols
Dulbecco’s phosphate-buffered saline, Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor Single-Use Cocktail, Pierce BCA Protein Assay Kit, and Trypsin/EDTA solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Amphotericin B, penicillin G, and RIPA Buffer were purchased from Sigma-Aldrich Inc. (St. Luis, MO, USA). NC-Slides A8, Solution 3, and Via1-Cassettes were obtained from ChemoMetec (Lillerød, Denmark). Growth media (DMEM and RPMI 1640) and fetal bovine serum were acquired from CytoGen (Zgierz, Poland). Neomycin sulfate was obtained from 1Amara (Kraków, Poland). The remaining chemicals were purchased from POCH S.A. (Gliwice, Poland).
Marciniec K., Rzepka Z., Chrobak E., Boryczka S., Latocha M., Wrześniok D, & Beberok A. (2023). Design, Synthesis and Biological Evaluation of Quinoline-8-Sulfonamides as Inhibitors of the Tumor Cell-Specific M2 Isoform of Pyruvate Kinase: Preliminary Study. Molecules, 28(6), 2509.
+ Open protocol
+ Expand
8

Protein Quantification in Myotube Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols
Myotubes were lyzed in RIPA buffer (Merck-Millipore, VIC) with 1 μL/mL protease inhibitor cocktail (Sigma-Aldrich) and 10 μL/mL Halt Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific, Rockford, IL, United States). Protein concentrations were determined using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific) according to the manufacturer’s protocol, and absorbance was measured at 562 nm on a Synergy 2 Microplate Reader (BioTek, Winooski, VT, United States).
Brown E.L., Foletta V.C., Wright C.R., Sepulveda P.V., Konstantopoulos N., Sanigorski A., Della Gatta P., Cameron-Smith D., Kralli A, & Russell A.P. (2018). PGC-1α and PGC-1β Increase Protein Synthesis via ERRα in C2C12 Myotubes. Frontiers in Physiology, 9, 1336.
+ Open protocol
+ Expand
9

Cell Lysis Protocol for Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
In general, cells were seeded in 12-well plates and lysed at the indicated time points in 30 μl lysis buffer (100 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.5% Nonidet P-40, cOmplete protease inhibitor (Roche) and Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific)). The cell lysates were processed as described by Eichwald et al., 2004 [24 (link)].
Glück S., Buttafuoco A., Meier A.F., Arnoldi F., Vogt B., Schraner E.M., Ackermann M, & Eichwald C. (2017). Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle. PLoS ONE, 12(6), e0179607.
+ Open protocol
+ Expand
10

Western Blot Protocol for Protein Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were lysed in RIPA Lysis and Extraction Buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche, catalog no. 04693124001) and Halt Phosphatase Inhibitor Single-Use Cocktail (Thermo Fisher Scientific, catalog no. 78428). Protein extracts were then separated by electrophoresis on a 4%–15% precast SDS polyacrylamide gel (Bio-Rad, catalog no. 4561084 or 4561086). Separated proteins in the gel were then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, catalog no. 1620177) under 100 V for 75 minutes. After transfer, blot was blocked with 5% nonfat dry milk in TBS with 0.1% Tween 20 detergent (TBST) and then stained with the primary antibody. Horseradish peroxidase–conjugated secondary antibodies were used for all Western blots. Signals were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, catalog no. 32106). Information of the primary antibodies is included in Supplementary Table S2.
Wan Y., Zhao Y., Cao M., Wang J., Tran S.V., Song Z., Hsueh B.W, & Wang S.E. (2024). Lung Fibroblasts Take up Breast Cancer Cell-derived Extracellular Vesicles Partially Through MEK2-dependent Macropinocytosis. Cancer Research Communications, 4(1), 170-181.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!