Phosphosafe

Manufactured by Merck Group

Sourced in Germany, United States

PhosphoSafe is a laboratory reagent used to preserve the phosphorylation state of proteins during cell lysis and sample preparation. It acts as a powerful phosphatase inhibitor, preventing the dephosphorylation of proteins and enabling the accurate analysis of their phosphorylation status.

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Lab products found in correlation

Bradford assay, by Bio-Rad (1 mentions) Luminex xmap magpix, by Merck Group (1 mentions) Magpix xponent software, by Merck Group (1 mentions) Proliferating cell nuclear antigen (pcna), by Merck Group (1 mentions) β actin, by Beyotime (1 mentions) Bm chemiluminescence blotting substrate, by Roche (1 mentions) Ecl plus kit, by GE Healthcare (1 mentions) Horseradish peroxidase coupled secondary antibody, by Abcam (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Vimentin, by Merck Group (1 mentions) Ibright fl1000, by Thermo Fisher Scientific (1 mentions) Polyclonal antibodies, by Abcam (1 mentions) Protease inhibitor, by Roche (1 mentions) Il 1β, by R&D Systems (1 mentions) Interleukin 6 (il 6), by RayBiotech (1 mentions) Tnf α, by RayBiotech (1 mentions) Bio plex 200, by Bio-Rad (1 mentions) Procartaplex human kits, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Anti β actin, by Merck Group (1 mentions)

Spelling variants of this product

PhosphoSafe™ Extraction Reagent (56 citations) PhosphoSafe extraction buffer (15 citations) PhosphoSafe buffer (14 citations) PhosphoSafe lysis buffer (12 citations)

7 protocols using phosphosafe

Quantifying Ocular Inflammatory Mediators

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The intact right eyes of all the studied groups were macerated in liquid nitrogen and placed in eppendorfs, which were added with 500 μL of protease (Protease Inhibitor Cocktail Set I, Cat. No. 53391, Millipore Corporation, CA, USA) and phosphatase (PhosphoSafe, Cat. No. 7,126-3-3, Novagen, Millipore Corporation, Billerica, CA, USA) inhibitor solution prepared according to the manufacturer’s instructions. The material was incubated for 20 min at 4 °C under constant stirring and centrifuged at 14,000 RPM for 10 min at 4 °C. The supernatants were then collected and immediately frozen at −80 °C. The protein concentration in the supernatant was measured using a Bradford assay (Bio-Rad, Hemel Hempstead, UK).
IL-1β, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and TNF-α inflammatory mediators were quantified in the eye macerate supernatant and in blood plasma using the rat cytokine MILLIPLEX MAP Kit (RECYTMAG-65K; Millipore Corporation, Billerica, CA, USA) according to the manufacturer’s instructions and analyzed on the LUMINEX xMAP MAGPIX (Millipore Corporation, Billerica, CA, USA) equipment. The concentration of analytes was determined by MAGPIX xPONENT software (Millipore Corporation, Billerica, CA, USA). Results were expressed as mean ± SEM of cytokine concentrations (pg/mL).

Girol A.P., de Freitas Zanon C., Caruso Í.P., de Souza Costa S., Souza H.R., Cornélio M.L, & Oliani S.M. (2021). Annexin A1 Mimetic Peptide and Piperlongumine: Anti-Inflammatory Profiles in Endotoxin-Induced Uveitis. Cells, 10(11), 3170.

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Protein Expression Analysis in SSCs

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Total cell extracts were prepared from SSCs cultured in control medium or media containing different concentration of PD0325901. Total proteins were resolved by PhosphoSafe (Millipore), and western blotting analysis was performed according to the protocol we described previously (Cao et al. 2011 (link), Yu et al. 2014) . Briefly, proteins were transferred to PVDF membrane and incubated with antibodies including b-actin (1:1000, Beyotime, Haimen, Jiangsu, China), PCNA (1:2000, Millipore), c-MYC (1:2500, Chemicon), ERK1/2 (1:1000, C.S.T), pERK1/2 (1:1000, C.S.T) at 4 8C for overnight. The next day, the secondary antibody were added, and incubated. Then, the detection was performed using the BM-chemiluminescence blotting substrate (Roche) (Yu et al. 2014 (link), Zhang et al. 2011) (link).

Niu Z., Zheng L., Wu S., Mu H., Ma F., Song W., Zhu H., Wu J., He X, & Hua J. (2015). Ras/ERK1/2 pathway regulates the self-renewal of dairy goat spermatogonia stem cells. Reproduction (Cambridge, England), 149(5).

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Quantification of VEGFA Protein Expression

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Cells were lysed in PhosphoSafe (MERCK Life Sciences, Billerica, MA, USA). Equal amounts of protein isolates were separated on SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). For VEGFA protein expression, we used 1 mg/mL polyclonal antibodies (Abcam, Cambridge, UK). Vimentin (Sigma-Aldrich, Saint Louis, MS, USA) served as loading control. For visualization, we used horseradish peroxidase-coupled secondary antibodies (Abcam and Agilent) and the ECL Plus kit (GE Healthcare, Chalfont St. Giles, UK). iBright FL1000 (Thermo Fisher, Waltham, MS, USA) was used for processing and visualization. All western blots were repeated three times showing comparable results.

Krebs M., Kotlyar M.J., Fahl J., Janaki Raman S., Röhrig F., Marquardt A., Kübler H., Kneitz B., Schulze A, & Kalogirou C. (2023). Metformin Regulates the miR-205/VEGFA Axis in Renal Cell Carcinoma Cells: Exploring a Clinical Synergism with Tyrosine Kinase Inhibitors. Urologia Internationalis, 108(1), 49-59.

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Extracting and Analyzing Hippocampal Proteins

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Protein extraction and analysis was done as described
previously.^{44 (link)} Mice
were decapitated; the hippocampi were taken out, immediately frozen on
dry ice and stored at –80°C. For the analysis of myelin and
control proteins, samples were homogenized in lysis buffer
(50 mM Tris HCl (pH 8.3), 150 mMNaCl, 40 mM NaF, 5 mM EDTA,
5 mM EGTA, 1 mMNa₃VO₄, 1% Igepal, 0.1% sodium
deoxycholate, 0.1% sodium dodecyl sulfate) also containing
1 mM phenylmethysulfonylfluoride,
10 μg ml^–1 aprotinin and
10 μg ml^–1 leupeptin. The tissue
lysates were freeze-thawed four times and homogenized by pulling through
a 1 ml syringe 10 times and centrifuged at
12 000 r.p.m. for 45 min. To estimate the
phosphorylation of pMAPK/MAPK, total protein was extracted from
cultures by using PhosphoSafe (Merck) according to the
manufacturer's instructions.

Hassouna I., Ott C., Wüstefeld L., Offen N., Neher R.A., Mitkovski M., Winkler D., Sperling S., Fries L., Goebbels S., Vreja I.C., Hagemeyer N., Dittrich M., Rossetti M.F., Kröhnert K., Hannke K., Boretius S., Zeug A., Höschen C., Dandekar T., Dere E., Neher E., Rizzoli S.O., Nave K.A., Sirén A.L, & Ehrenreich H. (2016). Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus. Molecular Psychiatry, 21(12), 1752-1767.

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Protein Profiling of Nerve Tissues

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For protein detection, dissected nerve tissues were chopped, spinal cord samples were pottered, and afterwards, DRG, spinal cord and nerve samples were sonicated (2 × 60%, 10 s) in 100–500 µL of, respectively, cell lysis buffer, a mixture of phosphosafe (Merck, Darmstadt, Germany) and protease-inhibitor (Roche Holding AG, Basel, Switzerland) or ice-cold PBS (R&D systems, Minneapolis, MN, USA/Ray Biotech, Peachtree Corners, GA, USA). Afterwards, the samples were centrifuged for 10 min at 10,000 rpm. The supernatant was then used for Bradford (Sigma-Aldrich) measurements, as described previously (Bradford 1976), followed by IL-1β (R&D systems, Minneapolis, Minnesota, USA), IL-6, TNFα (Ray Biotech), NGF (DLDevelop, Wuxi, Jiangsu, China), ELISA or Luminex Multiplex measurements (Invitrogen, Carlsbad, CA, USA). In the Luminex Multiplex measurement, the following cyto- and chemo-kines were measured: FGFβ, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p40/p70), IL-13, Il-17, IP-10, KC, MCP-1, MIG, MIP-1α, TNF-α and VEGF. The different ELISAs and the Luminex Multiplex were performed according to the manufacturer’s description. All samples were measured in duplicates. For calculation of the protein concentrations, Graph Pad Prism 7 was used.

Osthues T., Zimmer B., Rimola V., Klann K., Schilling K., Mathoor P., Angioni C., Weigert A., Geisslinger G., Münch C., Scholich K, & Sisignano M. (2020). The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain. Cells, 9(7), 1740.

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Cytokine and Chemokine Profiling in Tumor and Nerve Tissue

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Cytokine and chemokine levels were determined in tumors and the sciatic nerve using the Mouse Cytokine/Chemokine bead immunoassay kit, (ProcartaPlex Human kits, eBioscience, San Diego, CA, USA). Tissue samples were frozen directly at −80 °C until they were used for LUMINEX measurement. Nerves and tumors were lysed in 400 µL lysis buffer (50% PhosphoSafe and 50% Protease inhibitor cocktail (Merck, Darmstadt, Germany). Samples were cut in small pieces and then sonicated once at 60% for 10 s. Afterwards all samples were centrifuged for 10 min at 10.000× g. In 50 µL of the samples the concentrations of VEGF-A, IFN-gamma, IL-1alpha, IL-1beta, IL-10, IL-4, IL-5, IL-6, IL-9, Leptin, M-CSF, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha and MCP-1/CCL2 were determined using a Bioplex 200 (BioRad, Hercules, CA, USA).

Cohnen J., Kornstädt L., Hahnefeld L., Ferreiros N., Pierre S., Koehl U., Deller T., Geisslinger G, & Scholich K. (2020). Tumors Provoke Inflammation and Perineural Microlesions at Adjacent Peripheral Nerves. Cells, 9(2), 320.

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Western Blot Analysis of EMT Markers

Cited 1 time

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Cells were lysed in either RIPA or PhosphoSafe buffer (Calbiochem/Merck Millipore, Darmstadt, Germany) at 70–90% confluency and at different time points during continuous culture. Equal amounts of cellular proteins were fractionated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted as described in detail earlier [38 (link)]. The antibodies used were anti-E-cadherin (#610181) and anti-Rac1 (#610650), BD Transduction Laboratories (Heidelberg, Germany); anti-Rac1b, (#09-271), Merck Millipore; anti-Vimentin, anti-GAPDH (14C10), #2118, Cell Signaling Technology (CST, Frankfurt am Main, Germany); anti-β-actin, Sigma (Deisenhofen, Germany); anti-Claudin-4, Clone 3E2C1, #18-7341, Zymed Laboratories (South San Francisco, CA, USA); anti-Snail, #3895, CST; anti-HSP90 (F-8), #sc-13119, Santa Cruz Biotechnology (Heidelberg, Germany). The HRP-linked anti-rabbit, #7074, and anti-mouse, #7076, secondary antibodies were from CST. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany) were used for chemiluminescent detection of proteins on a BioRad ChemiDoc XRS imaging system. This device also allowed for the densitometric quantification of signal intensities from underexposed autoradiographs. The original Western blot images can be found at Figure S5–S10.

Schmidtlein P.M., Volz C., Braun R., Thürling I., Lapshyna O., Wellner U.F., Konukiewitz B., Lehnert H., Marquardt J.U, & Ungefroren H. (2021). A Comparative Endocrine Trans-Differentiation Approach to Pancreatic Ductal Adenocarcinoma Cells with Different EMT Phenotypes Identifies Quasi-Mesenchymal Tumor Cells as Those with Highest Plasticity. Cancers, 13(18), 4663.

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