Halt protease phosphatase inhibitor

Manufactured by Thermo Fisher Scientific

Sourced in United States

Halt Protease/Phosphatase Inhibitor is a broad-spectrum inhibitor that effectively suppresses the activity of proteases and phosphatases in biological samples. It is designed to preserve the integrity of proteins and prevent degradation during sample preparation and analysis.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Halt™ Protease & Phosphatase Inhibitors Cocktail (3 citations) HALT phosphatase/protease inhibitors (2 citations) Halt’s protease and phosphatase inhibitors (3 citations) Halt protease and phosphatase inhibitors cocktail (19 citations) Halt protease inhibitor (338 citations) Halt phosphatase inhibitor (97 citations) Protease/phosphatase inhibitor (97 citations) Halt Protease (66 citations) Phosphatase Halt (2 citations) HALT inhibitors (5 citations)

63 protocols using halt protease phosphatase inhibitor

Detergent-Based Protein Fractionation Assay

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For the solubility assays, cell lysis and protein fractionation based on detergent solubility were performed as previously described (Figure 2A; Guo and Lee, 2011 (link); Kfoury et al., 2012 (link); Silva et al., 2016 (link)). Briefly, higher solubility proteins (S fractions) were purified in 1% Triton buffer [1% Triton X-100 (Thermo Fisher Scientific), 1% Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientific), 1:5,000 Benzonase (Sigma) and 10 mM DTT (New England BioLabs) in DPBS], whereas lower solubility pelleted proteins (P fractions) were resuspended in 5% SDS buffer [5% SDS (Sigma), 1% Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientific), 1:5,000 Benzonase (Sigma) and 10 mM DTT (New England BioLabs) in RIPA buffer]. SDS-PAGE western blot was performed by loading 20 μg of each S-fraction and equal volume of the P-fraction onto pre-cast Tris-Acetate SDS-PAGE (Novex, Invitrogen). Western blot was performed as before. Densitometry values (pixel mean intensity in arbitrary units, a.u.) were measured with the Histogram function of Adobe Photoshop 2021 (v.22.4.3) and normalized to the respective GAPDH intensity in the S-fraction. Calculations were done in Microsoft Excel (v.16.52) and graphs were plotted in GraphPad Prism 9 (v.9.2.0).

Silva M.C., Nandi G., Donovan K.A., Cai Q., Berry B.C., Nowak R.P., Fischer E.S., Gray N.S., Ferguson F.M, & Haggarty S.J. (2022). Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy. Frontiers in Cellular Neuroscience, 16, 801179.

+ Open protocol

+ Expand

Murine Tissue Harvesting and Serum Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

After in vivo experiments animals were humanely killed by CO₂ asphyxiation in a closed chamber and tissues (bladder, prostate, and prostatic urethra) dissected for histological or molecular assays. Blood samples were obtained through cardiac punctures of mice under deep isoflurane anaesthesia using a 20-gauge needle and 3 ml syringe. Blood was allowed to coagulate in uncoated collection tubes at room temperature then centrifuged (1500 × g, 10 min, 4° C) to collect the serum. Tissue samples for molecular studies were snap-frozen on dry ice with isopropyl alcohol immediately following dissection. Protein lysates for western blot were obtained by bead homogenisation of tissue samples in Ca²⁺- and Mg²⁺-free Hank’s buffered saline solution with HaltTM protease/phosphatase inhibitor (87786, Thermofisher Scientific, Pittsburgh, PA, USA). Protein concentrations were determined by BCA protein assay (23252, Thermofisher) and lysates were snap-frozen until used for assays. The cGMP (ab65356, Abcam, Waltham, MA, USA) and serum testosterone (582701, Cayman Chemicals, Ann Arbor, MI, USA) ELISAs were performed following the manufacturers’ recommendations.

Zabbarova I.V., Ikeda Y., Kozlowski M.G., Tyagi P., Birder L., Chakrabarty B., Perera S., Dhir R., Straub A.C., Sandner P., Andersson K.E., Drake M., Fry C.H, & Kanai A. (2022). Benign prostatic hyperplasia/obstruction ameliorated using a soluble guanylate cyclase activator. The Journal of pathology, 256(4), 442-454.

+ Open protocol

+ Expand

Western Blotting and Immunoprecipitation Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

For western blotting, the cells and patient samples were lysed in RIPA buffer (Millipore, Billerica, Massachusetts) with Halt^TM protease & phosphatase inhibitor (Thermo Scientific, Rockford, IL). For immunoprecipitation, the primary antibodies (Gab1, 1:50 and rictor, 1:50) were incubated with Dynabeads^® Protein G (Invitrogen Dynal AS, Oslo, Norway) for 1 h at 25°C, followed by incubation with the cell lysates (500 μg-4 mg) for 2 h at 25°C. Total cell extracts and immunoprecipitated proteins were fractionated by SDS-PAGE followed by immunoblotting. The blots were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL) and detected using X-ray film (Fuji Photo Film, Tokyo). The quantitation of the western blot was quantified by Multi Gauge V3.0. The experiments were repeated in triplicate.

Chang C.H., Chan P.C., Li J.R., Chen C.J., Shieh J.J., Fu Y.C., Chen H.C, & Wu M.J. (2015). Gab1 is essential for membrane translocation, activity and integrity of mTORCs after EGF stimulation in urothelial cell carcinoma. Oncotarget, 6(3), 1478-1489.

+ Open protocol

+ Expand

Mitochondrial Isolation from Liver Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Liver tissue is homogenized with Potter-Elvehjem glass homogenizer (size 22: 100–150 µm clearance; (Kimble Chase, Rockwood, TN, USA) in with 5 mL ice-cold mitochondria isolation buffer (0.2 mM EDTA, 0.25 M sucrose, 10 mM Tris-HCl pH 7.8) supplemented with 1× Halt^TM Protease & Phosphatase Inhibitor (Thermo Fisher Scientific, Darmstadt, Germany). Homogenates were centrifuged at 1000 g for 10 min at 4 °C to pellet the cells debris and nuclei. The supernatant was centrifuged again at 1000 g for 10 min at 4 °C to remove remaining debris. The supernatant was then centrifuged at 10,000 g for 15 min at 4 °C to pellet the mitochondrial fraction. The pellet was washed in fresh mitochondria-isolation buffer and pelleted again by centrifugation at 10,000 g for 15 min at 4 °C. Finally, mitochondrial enriched pellet was suspended in mitochondria isolation buffer, and protein concentration was determined by BCA assay according to standard procedures (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Mannheim, Germany).

Schilf P., Künstner A., Olbrich M., Waschina S., Fuchs B., Galuska C.E., Braun A., Neuschütz K., Seutter M., Bieber K., Hellberg L., Sina C., Laskay T., Rupp J., Ludwig R.J., Zillikens D., Busch H., Sadik C.D., Hirose M, & Ibrahim S.M. (2021). A Mitochondrial Polymorphism Alters Immune Cell Metabolism and Protects Mice from Skin Inflammation. International Journal of Molecular Sciences, 22(3), 1006.

+ Open protocol

+ Expand

Platelet activation by sickle hemoglobin

Check if the same lab product or an alternative is used in the 5 most similar protocols

Washed platelets were incubated with various concentrations of HbS. Platelet pellet was washed and lysed with RIPA buffer in presence of Halt^TM protease-phosphatase inhibitor (Thermo Scientific, USA). The lysis products were processed for SDS PAGE and immunoblotted for all above signaling molecules associated with platelet activation including Lyn, PI3K, Akt and ERK (phosphor and non-phospho).

Annarapu G.K., Singhal R., Gupta A., Chawla S., Batra H., Seth T, & Guchhait P. (2016). HbS Binding to GP1bα Activates Platelets in Sickle Cell Disease. PLoS ONE, 11(12), e0167899.

+ Open protocol

+ Expand

Liver Tissue Protein Extraction

Check if the same lab product or an alternative is used in the 5 most similar protocols

Fresh liver tissues (0.1 g) were homogenized in 900 μL T-PER TM Tissue Protein Extraction Reagent (Thermo) containing Halt TM Protease & Phosphatase Inhibitor (Thermo) using a rotor-stator homogenizer (Coyote G100) . The homogenates were centrifuged for 10 min at 12000 rpm/min at 4℃, with the supernatant gathered and stored at -20℃.

Wu T., Xie Y., Wu Z., Li Y., Jiang M., Yu H., Li X., Wang J., Zhou E, & Yang Z. (2023). β-Carotene Protects Mice against Lipopolysaccharide and D-Galactosamine Induced Acute Liver Injury via Regulation of NF-κB, MAPK, and Nrf2 Signaling. Journal of oleo science, 72(11).

+ Open protocol

+ Expand

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Fresh homogenized liver tissues, were lysed using protein extraction solution containing Halt TM Protease & Phosphatase Inhibitor (Thermo Fisher Scientific) followed by centrifugation for 10 min at 12000 rpm/min at 4℃. The supernatant was gathered, and the protein concentration was detected via a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific) . After quantification, those samples containing equal quantity of proteins, were separated with 10/12％ SDS-PAGE. Afterwards, proteins were transferred on Polyvinylidene fluoride (PVDF) membranes (Merck) , which were blocked in 5％ skim milk for 2 h at RT, incubated with specific primary antibody at 4ºC overnight, and probed with relative secondary antibody. In the end, protein bands on membranes were visualized with chemiluminescent substrates (Merck Millipore) by ECL Plus Western Blotting System.

+ Open protocol

+ Expand

Immunoprecipitation of PDGF-BB Receptor

Check if the same lab product or an alternative is used in the 5 most similar protocols

Co-IP was performed using the Dynabeads Co-Immunoprecipitation Kit (ThermoFisher Scientific, Waltham, MA, USA), using polyclonal goat anti-mouse PDGF-BB antibody (Novus NBP1-52533) for immunoprecipitation. Briefly, a whole brain from a P21 WT (c57BL/6, n = 1) male mouse was harvested and homogenized in immunoprecipitation buffer, following a thoracotomy and PBS perfusion. Dynabeads were coupled with 10 ug of antibody per mg of beads. Whole-brain lysate (1.5 g protein) was processed with 1.5 g coupled beads according to the manufacturer’s instruction, and using the following extraction buffer: 100 mM NaCl, EDTA-free HALT Protease\Phosphatase Inhibitor (ThermoFisher Scientific, Waltham, MA, USA), 2 mM MgCl₂, and 1 mM DTT. Aliquots were collected at each step for downstream analysis. Eluted protein was immunoblotted under denaturing conditions (see Western blotting protocol above), using 1:1000 polyclonal goat anti-mouse PDGFRβ (R&D, AF1042), and 1:2000 donkey anti-goat AlexaFluor488 (Jackson ImmunoResearch, West Grove, PA, USA, 705-545-147).

Payne L.B., Abdelazim H., Hoque M., Barnes A., Mironovova Z., Willi C.E., Darden J., Houk C., Sedovy M.W., Johnstone S.R, & Chappell J.C. (2023). A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging. Biomolecules, 13(4), 711.

+ Open protocol

+ Expand

Immunoblot Analysis of MAPK Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were plated to confluency in 6-well plastic cell culture plates and starved with 0.5% HS, DMEM/F12 overnight before specified treatment and lysate collection. Protein samples were collected in RIPA buffer (CST) with 1× HALT protease/phosphatase inhibitor (Thermo) and reduced in Laemlli SDS buffer (BioRad) with BME (Sigma). Protein was loaded onto 4–15% gradient polyacrylamide gels (Bio-Rad). After electrophoresis proteins were transferred to Trans-Blot nitrocellulose membranes (Bio-Rad, 1704159EDU). Blots were probed overnight at 4 °C with mouse MEK1/2 (CST 4694S), rabbit pMEK1/2 (CST 9154S), rabbit pMKK7 (Abcam ab4762), rabbit pMKK4 (CST 4514S), rabbit pMKK3/6 (CST9231), rabbit COT (Genetex GTX102711), TAK1 (SCBT), mouse anti-HSC70 (Santa Cruz Biotechnology), and IRDye donkey anti-rabbit 800 and goat anti-mouse 680 secondary antibodies (LiCor) before imaging. All images were acquired on an Odyssey Infrared Scanner (LiCor).

Peterson A.F., Ingram K., Huang E., Parksong J., McKenney C., Bever G.S, & Regot S. (2022). Systematic Analysis of the MAPK Signaling Network Reveals MAP3K Driven Control of Cell Fate. Cell systems, 13(11), 885-894.e4.

+ Open protocol

+ Expand

Western Blot Analysis of Tumor Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells treated under designated conditions were lysed using cell lysis buffer supplemented with Phosphatase and Protease Inhibitor cocktail (Sigma-Aldrich). For tumor tissue process, tumor samples were kept on dry ice at all times and a tissue pulverizer was dipped in liquid nitrogen before use. Tumor tissues were first pulverized and then lysed in cell lysis buffer supplemented with Halt Protease Phosphatase inhibitor (ThermoFisher, Waltham, Massachusetts, USA). Proteins were resolved in SDS-PAGE and transferred to PVDF membranes. After blockage with 5% BSA in TBX (1×) blocking buffer, the membranes were incubated with primary antibodies with 1% BSA in TBST overnight at 4°C and then blotted with HRP-conjugated secondary antibodies (Cell Signaling Technology). Immunoblotting detection was performed with GE ECL Prime reagent (GE Healthcare, Piscataway, New Jersey, USA) and images were captured using FuJi LAS 3000 system (FUJIFILM Corporation, Tokyo, Japan).

Yu Y., Hall T., Eathiraj S., Wick M.J., Schwartz B, & Abbadessa G. (2017). In-vitro and in-vivo combined effect of ARQ 092, an AKT inhibitor, with ARQ 087, a FGFR inhibitor. Anti-Cancer Drugs, 28(5), 503-513.

+ 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?

Revolutionizing how scientists
search and build protocols!