Phosphatase inhibitor cocktail

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Phosphatase inhibitor cocktail is a solution that contains a mixture of compounds designed to inhibit the activity of various phosphatases. Phosphatases are enzymes that remove phosphate groups from proteins, and their inhibition can be useful in various biochemical and cell-based research applications.

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Close spelling variants of this product

Phosphatase inhibitors (26 citations) Protease and phosphatase inhibitor cocktail (10 citations) Phosphatase Inhibitor Cocktail B (6 citations) Phosphatase inhibitor cocktail A (5 citations) Phosphatase Inhibitor cocktails A and B (4 citations) Phosphatase inhibitor cocktail C (3 citations) Proteinase inhibitor cocktail and phosphatase inhibitor (2 citations) Halt™ Phosphatase Inhibitor Cocktail (2 citations) Proteinase and phosphatase inhibitor cocktail (2 citations)

21 protocols using phosphatase inhibitor cocktail

Quantitative Immunohistochemical Analysis of Tumor Markers

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Cells were lysed in RIPA buffer (Sigma) with protease inhibitor cocktail
(Roche and phosphatase inhibitor cocktails (Santa Cruz Biotechnology) for
Western blotting. Paraffin-embedded formalin-fixed sections were subjected to
heat-induced antigen retrieval. The biotin-streptavidin system (Vector
Laboratories) and DAB were used to visualize human Ki-67, while Alexa Fluor
conjugates (Life Technologies) and TSA plus fluorescein system (Perkin Elmer)
were used to detect human PDGFRβ, EGFR, c-MET and mouse ki-67, PD-L2, CD8
and CD11c. For immuno-cytochemistry, cells were fixed with 4% PFA and stained
with Alexa Fluor 555 (for Ki-67) and Alexa Fluor 488 (for CD271) conjugates
(Life Technologies). Nuclei were counterstained by hematoxylin or DAPI.
Fluorescent images were digitized using the Applied Imaging Ariol SL-50scanner
at 20x magnification. Quantification of CD8 and Ki-67 signals was performed
using the Definiens Tissue Studio 4.3 (Definiens, Inc.) according to the
manufacturer’s instruction. Please refer to Supplementary Methods for primary
antibody information. Cell surface PD-L1 and PD-L2 were detected by flow
cytometry using APC-conjugated anti-PD-L1 and PE-conjugated anti-PD-L2
antibodies (BioLegend).

Song C., Piva M., Sun L., Hong A., Moriceau G., Kong X., Zhang H., Lomeli S., Qian J., Yu C.C., Damoiseaux R., Kelley M.C., Dahlman K.B., Scumpia P.O., Sosman J.A., Johnson D.B., Ribas A., Hugo W, & Lo R.S. (2017). Recurrent tumor cell-intrinsic and -extrinsic alterations during MAPKi-induced melanoma regression and early adaptation. Cancer discovery, 7(11), 1248-1265.

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Schwann Cell Protein Expression Analysis

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Schwann cells were cultured in low-glucose (1g/mL) DMEM (Sigma) containing 3% FBS and treated with 10 ng/mL of Tgfβ1, Tnc, Btc, or FGF5 for 24 hours, or with 50 nM cAMP for 24, 48, or 72 hours. Following treatment, cells were lysed in 200 µL of radio-immunoprecipitation assay buffer plus phosphatase inhibitor cocktails (1:100, Santa Cruz Biotechnology, Dallas, TX, USA, sc-45045 and sc-45065). Cell lysates were centrifuged at 16,000 × g for 15 minutes at 4°C. The supernatants were transferred to new 1.5-mL tubes, and protein concentrations were determined using a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Swindon, UK). A volume containing 20 µg of protein was then mixed with 4× sample buffer for electrophoresis. Nerves were harvested and directly sonicated in 1 × SDS loading buffer. Western blotting was performed as previously described (Woodley et al., 2019). ImageJ software (Version, 1.53r, National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012) was used to quantify the band intensity from three independent western blots.

Hu R., Dun X., Singh L, & Banton M.C. (2023). Runx2 regulates peripheral nerve regeneration to promote Schwann cell migration and re-myelination. Neural Regeneration Research, 19(7), 1575-1583.

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Protein and RNA Extraction Protocol

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Proteins were extracted from 2 x 10⁶ to 3 x 10⁶ cells, cells were pelleted at 300 x g for 5 minutes at room temperature. Pellets were washed with ice cold PBS then lysed using RIPA lysis buffer (Cell Signaling) supplemented with Phosphatase Inhibitor Cocktails (Santa Cruz Biotechnology). Lysates were centrifuged at 12000 x g for 15 minutes at 4°C and supernatant was kept at -80°C for further uses. Total RNA was isolated from cells using RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions and evaluated for quantity and quality by NanoDrop spectrophotometer.

Hasan Bou Issa L., Fléchon L., Laine W., Ouelkdite A., Gaggero S., Cozzani A., Tilmont R., Chauvet P., Gower N., Sklavenitis-Pistofidis R., Brinster C., Thuru X., Touil Y., Quesnel B., Mitra S., Ghobrial I.M., Kluza J, & Manier S. (2024). MYC dependency in GLS1 and NAMPT is a therapeutic vulnerability in multiple myeloma. iScience, 27(4), 109417.

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Western Blot Protocol for Protein Analysis

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Western blot was performed as described previously (37 (link)). The cells were collected in ice-cold PBS, and lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktail (Santa Cruz Biotechnology). The protein concentrations were determined by use of Bradford methods and boiled in SDS sample buffer (50 mM of Tris [pH 6.8]; 100 mM of DTT; 2% SDS; 0.1% bromophenol blue; 10% glycerol). The proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were then incubated in blocking solution (5% non-fat milk in 20 mM of TRIS-HCl, 150 mM of NaCl, 0.1% Tween-20) (TBST), followed by incubation with the indicated primary antibodies at 4°C overnight. Then secondary antibodies were incubated and detected with an enhanced chemiluminescence reaction.

Zhu X., Liu L., Wang Y., Cong J., Lin Z., Wang Y., Liu Q., Wang L., Yang B, & Li T. (2021). lncRNA MIAT/HMGB1 Axis Is Involved in Cisplatin Resistance via Regulating IL6-Mediated Activation of the JAK2/STAT3 Pathway in Nasopharyngeal Carcinoma. Frontiers in Oncology, 11, 651693.

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Macrophage Signaling Pathway Analysis

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Macrophages were lysed in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP40) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Santa Cruz Biotechnology). Cell lysates were then separated on a 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with the anti-phospho p44/42 MAPK (T202/Y204), anti-phospho p38, anti-p44/42, or anti-IκB antibodies (all from Cell Signaling Technology), and then followed by appropriate HRP-conjugated secondary Abs (Bio-Rad) and ECL reagent (Thermo Scientific). Macrophages were stimulated with COH1 WT or ΔSia mutant for 30 min at MOI = 10. Cells were then lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. Siglec-E was immunoprecipitated by mouse anti-Siglec-E mAb (generated in the Crocker lab). Immunoblots were probed with Ab to SHP-1 (Santa Cruz Biotechnology) and Siglec-E (R&D systems), respectively, and then followed by appropriate HRP-conjugated secondary Abs and ECL reagent.

Chang Y.C., Olson J., Beasley F.C., Tung C., Zhang J., Crocker P.R., Varki A, & Nizet V. (2014). Group B Streptococcus Engages an Inhibitory Siglec through Sialic Acid Mimicry to Blunt Innate Immune and Inflammatory Responses In Vivo. PLoS Pathogens, 10(1), e1003846.

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Co-Immunoprecipitation of LRP1 Protein

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To confirm protein-protein binding properties, co-immunoprecipitation (Co-IP) was performed as described previously [15] . Lysates of rat brain tissue were generated under addition of protease inhibitor cocktail and phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Texas, USA). The total protein of the lysates was measured by the Pierce BCA protein assay Kit (Thermo Scientific, Waltham, MA, USA) analyses by a GENESYS 10UV–VIS Spectrophotometer (Thermo Scientific, NY, USA). The rat brain proteins were used for Co-IP experiments performed with the Pierce Co-IP Kit (Thermo Scientific, Waltham, MA, USA). The Co-IP was done according to the manufacturer's protocol. Ten micrograms of the monoclonal LRP1 (Abcam, Cambridge, MA, USA) were incubated with the delivered resin and covalently coupled. The antibody-coupled resin was incubated with 200 ml of the whole rat testis protein lysates overnight at 4 °C. The resin was washed and the protein complexes bound to the antibody were eluted. Subsequent western blot analyses were performed as described before.

Peng J., Pang J., Huang L., Enkhjargal B., Zhang T., Mo J., Wu P., Xu W., Zuo Y., Peng J., Zuo G., Chen L., Tang J., Zhang J.H, & Jiang Y. (2019). LRP1 activation attenuates white matter injury by modulating microglial polarization through Shc1/PI3K/Akt pathway after subarachnoid hemorrhage in rats. Redox Biology, 21, 101121.

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Western Blot Analysis of Frozen EHTs

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The EHTs were flash frozen on dry ice and stored in −80°C freezer for up to 2 weeks. The proteins were homogenized in RIPA buffer supplemented with sodium orthovanadate, PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Santa Cruz Biotechnology). The protein was separated on precast 4%–20% Mini-PROTEAN TGXgels (Bio-Rad) before transfer to a PVDF membrane (Millipore). Samples were normalized using Revert Total Protein Stain (Li-Cor). The membrane was incubated in primary antibodies overnight at 4°C. The membranes were imaged on a Li-Cor Odyssey scanner. Quantification and analysis were performed using Image Studio Lite software (Li-Cor).

Shen S., Sewanan L.R., Shao S., Halder S.S., Stankey P., Li X, & Campbell S.G. (2022). Physiological calcium combined with electrical pacing accelerates maturation of human engineered heart tissue. Stem Cell Reports, 17(9), 2037-2049.

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Western Blotting for Protein Analysis

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Whole-cell lysates were prepared in RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail (Tech & Innovation^TM, Bucheon, Korea) and a phosphatase inhibitor cocktail (45065; Santa Cruz, CA, USA). Proteins were separated on an 8% or 10% SDS-PAGE gel, transferred to polyvinylidene fluoride (PVDF) membranes using an iBlot^TM dry blotting system (Invitrogen). Immunoblot analysis were performed with the following primary antibodies: pALK (Y1604) (1:1000, 3342; Cell Signaling Technology), ALK (1:1000, 3791; Cell Signaling Technology), p STAT3 (Y705) (1:1000, 9145; Cell Signaling Technology), STAT3 (1:1000, 9139; Cell Signaling Technology), pAKT (S473) (1:1000, 9271; Cell Signaling Technology), AKT (1:1000, 9272; Cell Signaling Technology), pERK (T202/Y204) (1:1000, 9101; Cell Signaling Technology), ERK (1:1000, 9102; Cell Signaling Technology), Acetyl-α-tubulin (Lys40) (1:1000, T7451; Sigma-Aldrich), GAPDH (1:2000, ab37168; abcam) and β-actin (1:10,000, 5441; Sigma-Aldrich, St. Louis, MO, USA) (Table S2). Secondary antibodies were rabbit or mouse horseradish peroxidase (HRP)-labelled secondary antibodies (1:10,000; Amersham, UK). The blots were visualised using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific-Pierce, Rockford, IL, USA). Source data from Western blot is shown in Figure S5.

Sampson J., Ju H.M., Song J.Y., Fry A.M., Bayliss R, & Choi J. (2022). A Polytherapy Strategy Using Vincristine and ALK Inhibitors to Sensitise EML4-ALK-Positive NSCLC. Cancers, 14(3), 779.

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Western Blot Analysis of Cellular Stress

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Cells or tissue sections were lysed in RIPA buffer (Sigma) with protease inhibitor (Roche) and phosphatase inhibitor cocktail (Santa Cruz). Samples containing 25 μg of protein were boiled in Laemmli buffer and resolved on 10% Tris-glycine polyacrylamide gels. Proteins were transferred to PVDF membranes, blocked with 5% milk and incubated overnight with primary antibodies as follow: mouse SMN (BD Pharmingen; 1:1000), rabbit ATF6α (Abcam ab37149; 1:500), mouse CHOP (Santa Cruz sc-7351; 1:100), rabbit GAPDH (Abcam ab9485; 1:2000), rabbit α-tubulin (Abcam ab126165; 1:2000). The blots were then incubated with respective HRP-conjugated antibodies at 1:2000 dilution.

Ng S.Y., Soh B.S., Rodriguez-Muela N., Hendrickson D.G., Price F., Rinn J.L, & Rubin L.L. (2015). Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy. Cell stem cell, 17(5), 569-584.

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Western Blot Protein Detection Protocol

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Cells were lysed with a Mammalian Protein Extraction Reagent lysis buffer (ThermoFisher Scientific, Waltham, Massachusetts, USA) containing a protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Santa Cruz Biotechnology, Dallas, Texas, USA). An amount of 15-30 µg proteins was cooked in the SDS loading buffer at 95 °C for 10 min, separated by SDS-gel electrophoresis and transferred to a nitrocellulose membrane (ThermoFisher Scientific, Waltham, MA, USA). Membranes were blocked for 1 hour at room temperature in TBST containing 10 % milk powder (Carl Roth, Karlsruhe, Germany) followed by overnight incubation with primary antibodies diluted in TBST at 4°C. For detection, horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Danvers, Massachusetts, USA) diluted in TBST containing 5 % milk powder were used in combination with ECL solution (Signal Fire ECL Reagent, Cell Signaling Technology, Frankfurt, Germany) to detect the bands via a Chemidoc XRS+ (BioRad, Hercules, California, USA).

Helfen A., Rieß J., Fehler O., Stölting M., An Z., Kocman V., Schnepel A., Geyer C., Gerwing M., Masthoff M., Vogl T., Höltke C., Roth J., Ng T., Wildgruber M, & Eisenblätter M. (2022). In vivo imaging of microenvironmental and anti-PD-L1-mediated dynamics in cancer using S100A8/S100A9 as an imaging biomarker. Neoplasia (New York, N.Y.), 28, 100792.

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