Halt protease and phosphatase inhibitor

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Japan, Germany

Halt protease and phosphatase inhibitors are lab equipment designed to inhibit the activity of proteases and phosphatases during protein extraction and analysis. They help maintain the integrity of target proteins by preventing unwanted degradation.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Protease inhibitors (1135 citations) Phosphatase inhibitor (390 citations) Halt protease inhibitor (338 citations) Halt phosphatase inhibitor (97 citations) Protease/phosphatase inhibitor (97 citations) Halt Protease (66 citations) Halt protease and phosphatase inhibitors cocktail (19 citations) HALT inhibitors (5 citations) HALTTM (3 citations) Halt’s protease and phosphatase inhibitors (3 citations)

334 protocols using halt protease and phosphatase inhibitor

Western Blot Analysis of Drug-Treated Cells

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

The DX tumor tissues were lysed with T-PER reagent (Thermo Fisher
Scientific, Waltham, MA USA) containing double concentration of Halt™
protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA USA).
For Western blot, cells were seeded on 6-well plates at density of 30,000 cells
per well in RPMI-1640 supplemented with 1% FBS, 2 mM L-glutamine, 25
mg/L Insulin and 100 mg/L penicillin/streptomycin. Next day, the cells were
treated with indicated drugs for 24 hours and lysed with RIPA lysis buffer
(Santa Cruz Biotechnology, Dallas, TX, USA) containing Halt™ protease
and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA USA). Protein
concentrations were measured using BSA assay. Western blot membranes were
scanned using Odyssey infrared imaging reagents including blocking buffer and
secondary antibodies (LI-COR, Lincoln, NE, USA).

Beglyarova N., Banina E., Zhou Y., Mukhamadeeva R., Andrianov G., Bobrov E., Lysenko E., Skobeleva N., Gabitova L., Restifo D., Pressman M., Serebriiskii I.G., Hoffman J.P., Paz K., Behrens D., Khazak V., Jablonski S.A., Golemis E.A., Weiner L.M, & Astsaturov I. (2016). Screening of conditionally reprogrammed patient-derived carcinoma cells identifies ERCC3-MYC interactions as a target in pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(24), 6153-6163.

+ Open protocol

+ Expand

Western Blot Analysis of PDX Tumors

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

Mouse and human PDX tumor tissues were lysed with T-PER reagent (78510, Thermo Scientific) containing double concentration of Halt™ protease and phosphatase inhibitors (78445, Thermo Scientific). For Western blot, KPC cells were seeded on 6-well plates at density of 30,000 cells per well in RPMI-1640 supplemented with 1% FBS, 2 mM L-glutamine, 25 mg/L Insulin and 100 mg/L penicillin/streptomycin. Next day, the cells were treated with indicated drugs for 24 hours and lysed with RIPA lysis buffer (Santa Cruz Biotechnology) containing Halt™ protease and phosphatase inhibitors (Thermo Scientific). Protein concentrations were measured using BSA assay. Western blot membranes were scanned using Odyssey infrared imaging reagents including blocking buffer and secondary antibodies (LI-COR).

Bobrov E., Skobeleva N., Restifo D., Beglyarova N., Cai K.Q., Handorf E., Campbell K., Proia D.A., Khazak V., Golemis E.A, & Astsaturov I. (2016). Targeted delivery of chemotherapy using HSP90 inhibitor drug conjugates is highly active against pancreatic cancer models. Oncotarget, 8(3), 4399-4409.

+ Open protocol

+ Expand

Assessing p53 Stability and Interactions

Cited 1 time

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

For SDS-resistance assay, log phase growing cells were pelleted and resuspended in cold Pierce IP Lysis Buffer (Thermo Fisher) freshly supplemented with 2× HALT protease and phosphatase inhibitor (Thermo Fisher). After incubation on ice for 30 minutes and cleared by centrifugation, 200 μg of native lysates were brought to a 500 μl volume in IP buffer and heated at 42°C in the presence of vehicle, SCR or 20 μM ReACp53 for 10 minutes, followed by a 30 minute incubation on ice. The protein samples were used for Immunoprecipitations with 5 μg of PAb240 antibody (Santa Cruz Biotech) at 4°C overnight, and analyzed with 15% SDS-PAGE gel electrophoresis and immunoblotting with anti-p53/DO-1 antibody[25 (link)].
For p53 stability, 200 μg of cell lysate in RIPA buffer was immunoprecipitated with 2 μg of anti-p53/DO-1 antibody. The immunoprecipitated complexes were purified with Pierce Direct Magnetic IP/Co-IP kit (Pierce) and analyzed with western blot.

Zhang Y., Xu L., Chang Y., Li Y., Butler W., Jin E., Wang A., Tao Y., Chen X., Liang C, & Huang J. (2019). Therapeutic potential of ReACp53 targeting mutant p53 protein in CRPC. Prostate cancer and prostatic diseases, 23(1), 160-171.

+ Open protocol

+ Expand

Assessing p53 Stability and Interactions

Cited 1 time

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

+ Open protocol

+ Expand

Mass Spectrometry Analysis of Drosophila Brain

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

MS/MS-LC data was previously described in (CORGIAT et al. 2021) . Briefly, ten biological replicates of 24 hr apf control (w 1118 ) or Nab2 ex3 pupal brains (60 brains per replicate) were lysed in urea buffer (8 M urea, 100 mM NaHPO4, pH 8.5) with HALT protease and phosphatase inhibitor (Pierce/Thermo Scientific) and processed at the Emory Proteomics Core. Separate samples were prepared for male and female brains. Label-free quantification analysis was adapted from a previously published procedure (SEYFRIED et al. 2017) (link). Data was analyzed using MaxQuant v1.5.2.8 with Thermo Foundation 2.0 for RAW file reading capability. Spectra were searched using the search engine Andromeda and integrated into MaxQuant against the Drosophila melanogaster Uniprot database (43,836 target sequences).
Analyses presented here used RStudio v1.1.453 (R-TEAM 2018), custom in-house scripts, and the following packages: ggpubr v0.2 (ALBOUKADEL 2018), cluster v2.1.0 (MAECHLER et al. 2016 ), and GOplot v1.0.2 (WALTER et al. 2015) , to examine 'planar cell polarity' annotated proteins. Gene ontology analyses were performed using FlyEnrichr (FlyEnrichr:amp.pharm.mssm.edu/FlyEnrichr/) (CHEN et al. 2013; (link)KULESHOV et al. 2016; (link)KULESHOV et al. 2019) (link), a Drosophila specific gene ontology enrichment analysis package.

Corgiat E.B., List S.M., Rounds J.C., Yu D., Chen P., Corbett A.H., & Moberg K.H. (2021). The Drosophila RNA binding protein Nab2 patterns dendritic arbors and axons via the planar cell polarity pathway.

+ Open protocol

+ Expand

Drosophila Proteome Analysis of Nab2 Mutant

Cited 3 times

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

MS/MS-LC data were previously described in Corgiat et al. (2021) (link). Briefly, 10 biological replicates of 24 h apf control (w¹¹¹⁸) or Nab2^ex3 pupal brains (60 brains per replicate) were lysed in urea buffer (8 M urea, 100 mM NaHPO4, pH 8.5) with HALT protease and phosphatase inhibitor (Pierce/Thermo Scientific) and processed at the Emory Proteomics Core. Separate samples were prepared for male and female brains. Label-free quantification analysis was adapted from a previously published procedure (Seyfried et al. 2017 (link)). Data were analyzed using MaxQuant v1.5.2.8 with Thermo Foundation 2.0 for RAW file reading capability. Spectra were searched using the search engine Andromeda and integrated into MaxQuant against the Drosophila melanogaster Uniprot database (43,836 target sequences). Analyses presented here used RStudio v1.1.453 (R Development Core Team 2018 ), custom in-house scripts, and the following packages: ggpubr v0.2 (Alboukadel 2018 ), cluster v2.1.0 (Maechler et al. 2016 ), and GOplot v1.0.2 (Walter et al. 2015 (link)), to examine “planar cell polarity” annotated proteins. GO analyses were performed using FlyEnrichr (FlyEnrichr: amp.pharm.mssm.edu/FlyEnrichr/; Chen et al. 2013 (link); Kuleshov et al.2016 (link), 2019 (link)), a Drosophila specific GO enrichment analysis package.

Corgiat E.B., List S.M., Rounds J.C., Yu D., Chen P., Corbett A.H, & Moberg K.H. (2022). The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism. G3: Genes|Genomes|Genetics, 12(6), jkac100.

+ Open protocol

+ Expand

Protein Extraction from Doxycycline-Treated Cells

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

Cells treated with doxycycline for specific times were removed from the incubator, collected on ice, pelleted and washed with cold (4°C) PBS. The pellet was resuspended in RIPA buffer (1X PBS, 1% Nonidet P-40, 0.5% Sodium Dodecyl Sulfate (SDS)) with 1:100 Halt Protease and Phosphatase inhibitor (Pierce Thermo Scientific)). Protein concentration was determined using BioRad's DC Protein Assay according to manufacturer's instructions. Samples were stored at −80°C.

Alexander-Savino C.V., Hayden M.S., Richardson C., Zhao J, & Poligone B. (2016). Doxycycline is an NF-κB inhibitor that induces apoptotic cell death in malignant T-cells. Oncotarget, 7(46), 75954-75967.

+ Open protocol

+ Expand

Quantifying Cellular Protein Extraction

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

Cellular or nuclear and cytoplasmic proteins were extracted using RIPA buffer (Nacalai Tesque, Kyoto, Japan) or NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Life Technologies) containing 1% Halt protease and phosphatase inhibitor (Life Technologies). Protein concentrations were determined using Pierce BCA Protein Assay Kits (Life Technologies). Amounts of TF and TFPI expression and NFκB (p65) activity in THP‐1 macrophages were measured using Quantikine ELISA kits for human TF and TFPI (R&D Systems), and NFκB (p65) Transcription Factor Assay Kits (ab133112; Abcam), respectively.

Watanabe Y., Koyama S., Yamashita A., Matsuura Y., Nishihira K., Kitamura K, & Asada Y. (2018). Indoleamine 2,3‐dioxygenase 1 in coronary atherosclerotic plaque enhances tissue factor expression in activated macrophages. Research and Practice in Thrombosis and Haemostasis, 2(4), 726-735.

+ Open protocol

+ Expand

Cytokine and Gene Expression Analysis of Bone Marrow-Derived Macrophages Treated with GCP-rCpa1

Cited 1 time

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

BMMs were incubated with GCP-rCpa1 (1:50 ratio) or treated with PBS as a control for 12 and 24 hours for qRT-PCR and cytokine analyses, respectively. Complementary DNA samples were synthesized from 1 µg of total RNA isolated from each sample of BMMs using oligo(dT) primer and SuperScript III™ Reverse Transcriptase (Invitrogen). The cDNA samples were used for qRT-PCR using Sybr-green detection method with gene specific primers. Mouse β-actin gene expression was used as an internal control. Results of real-time PCR data were derived from comparative C_t methods to detect relative gene expression as described (32 (link)). For cytokine assays, BMMs were spun down at 3,500 RPM for 3 mins and supernatant samples were stored in −80°C with Halt™ Protease and Phosphatase Inhibitor (Life Technologies). Cytokine concentrations were measured using Bio-Rad’s Bio-Plex Mouse cytokine 23-plex (catalog #:M60009RDPD) as described (27 (link)).

Campuzano A., Zhang H., Ostroff G.R., Dias L.D., Wüthrich M., Klein B.S., Yu J.J., Lara H.H., Lopez-Ribot J.L, & Hung C.Y. (2020). CARD9-associated Dectin-1 and Dectin-2 are Required for Protective Immunity of a Multivalent Vaccine Against Coccidioides posadasii Infection. Journal of immunology (Baltimore, Md. : 1950), 204(12), 3296-3306.

+ Open protocol

+ Expand

HEK-293 Cell Lysis and Protein Extraction

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

HEK-293 cells (CRL-1573, ATCC) were grown in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) at 37 °C in a humidified atmosphere with 5% CO₂. Cells were harvested, washed twice in phosphate-buffered saline, and frozen at 80 °C until used. Frozen cell pellets were lysed on ice in 8 m urea supplemented with 1× HALT protease and phosphatase inhibitor (Life Technologies). Lysates were clarified by centrifugation at 10,000 × g for 10 min at 4 °C and desalted with a Sep-Pak C18 1cc Vac Cartridge (100 mg, 55–105 μm particle size (Waters, Milford, MA). Protein in the lysates was measured with the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL).

Morales-Betanzos C.A., Lee H., Gonzalez Ericsson P.I., Balko J.M., Johnson D.B., Zimmerman L.J, & Liebler D.C. (2017). Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma. Molecular & Cellular Proteomics : MCP, 16(10), 1705-1717.

+ 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!