Phenyl methyl sulfonyl fluoride (pmsf)

Manufactured by MP Biomedicals

Sourced in United States

PMSF is a serine protease inhibitor used in biochemical applications to prevent the degradation of proteins by proteases. It irreversibly inhibits serine proteases such as trypsin, chymotrypsin, and plasmin.

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Lab products found in correlation

Sodium chloride (nacl), by MP Biomedicals (4 mentions) Protease inhibitor cocktail, by Roche (4 mentions) Mgcl2, by MP Biomedicals (4 mentions) Ethylene glycol tetraacetic acid (egta), by MP Biomedicals (4 mentions) Carboxy h2dcfda, by Thermo Fisher Scientific (4 mentions) Glutaraldehyde, by Ted Pella (4 mentions) Coomassie brilliant blue g 250, by Serva Electrophoresis (4 mentions) Trypsin, by Promega (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions) Ammonium acetate, by MP Biomedicals (3 mentions) Na2hpo4, by MP Biomedicals (3 mentions) Heparin, by MP Biomedicals (3 mentions) Potassium chloride (kcl), by MP Biomedicals (3 mentions) Ampicillin, by MP Biomedicals (3 mentions) Kh2po4, by MP Biomedicals (3 mentions) Cell lysis buffer, by Cell Signaling Technology (3 mentions) Advanced dmem f12 media, by Thermo Fisher Scientific (2 mentions) Pensterp cocktail, by Thermo Fisher Scientific (2 mentions) Dimethyl sulfoxide (dmso), by MP Biomedicals (2 mentions)

19 protocols using phenyl methyl sulfonyl fluoride (pmsf)

Detailed Biomolecular Reagents Protocol

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MES (M3671), BSA (82516), BES (14853), BCA (B9643), CuSO 4 (C2284) ThS (T1892), Toluidine Blue (T3260), MTT (M2128) were purchased from Sigma. IPTG (420322) and DTT (3870) were purchased from Calbiochem. Other chemicals such as Ampicillin (2007081), NaCl (194848), KCl (194844) , Na 2 HPO 4 (191437), KH 2 PO 4 (19142) , EGTA (194823) , MgCl 2 (191421) , PMSF (195381) , Ammonium acetate (191404) and Heparin (904108), DMSO were from purchased from MP Biomedicals and protease inhibitor cocktail was from Roche. Copper coated carbon grids were purchased from Ted Pella (01814F, carbon type-B, 400 mesh, Cu) DMEM advanced F12 media (12634010), Fetal Bovine serum (16000044), Pensterp cocktail (04693159001), Anti-anti (15240062) were purchased from Gibco.

Dubey T., Gorantla N.V., & Chinnathambi S. (2020). Photo-excited Toluidine Blue disaggregates the Repeat Tau and modulates cytoskeletal structure in neuronal cells.

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Biochemical Assays and Cell Culture Reagents

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MES (M3671), BSA (9048-46-8), BES (10191-18-1), BCA (B9643), CuSO4 (C2284), ThS (T1892), MTT (M2128) and Rose Bengal (330000) were purchased from Sigma. IPTG (420322) and DTT (3870) were purchased from Calbiochem. Other chemicals such as Ampicillin (2007081), NaCl (194848), KCl (194844), Na₂HPO₄ (191437), KH₂PO₄ (19142), EGTA (194823), MgCl₂ (191421), PMSF (195381), Ammonium acetate (191404), Heparin (904108) and DMSO were from purchased from MP biomedicals and protease inhibitor cocktail was from Roche (11697498001). Copper coated carbon grids were purchased from Ted Pella (01814.F, carbon type-B, 400 mesh Cu), DMEM advanced F12 media (12634010), Fetal Bovine Serum (16000044), Pensterp cocktail (04693159001) and Anti-anti (15240062) were purchased from Gibco. Tubulin (Thermo PA1-41331), actin (Thermo MA5-15739) were procured from thermo, total Tau antibody K9JA was purchased from Dako (K9JA, Dako A0024). The secondary antibodies Alexa Fluor 488 (A11034) and Alexa Fluor 555 (A32727) were purchased from thermo.

Dubey T., Gorantla N.V., Chandrashekara K.T, & Chinnathambi S. (2020). Photodynamic exposure of Rose-Bengal inhibits Tau aggregation and modulates cytoskeletal network in neuronal cells. Scientific Reports, 10, 12380.

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Western Blot Analysis of PRL-3 Signaling

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Cells were harvested by scraping into ice‐cold RIPA buffer containing PMSF (MP Biomedicals, Solon, OH, USA). The protein concentration was measured with a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) following the manufacturer's instructions. Western blots were performed as described previously 23, using antibodies specific for PRL‐3, ERK (extracellular signal‐regulated kinase) 1/2, phosphorylated ERK1/2 (pERK1/2), Slug, E‐cadherin, vimentin, and using GAPDH as control (Cell Signaling Technology, Danvers, MA, USA).

Dong Q., Ding X., Chang B., Wang H, & Wang A. (2015). PRL‐3 promotes migration and invasion and is associated with poor prognosis in salivary adenoid cystic carcinoma. Journal of Oral Pathology & Medicine, 45(2), 111-118.

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Immunoblotting Analysis of Uterine Samples

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Each uterine sample was prepared from a single uterine horn of one mouse. The uterine horn was cut into small pieces and lysed in a radioimmunoprecipitation assay buffer: 10 mM Tris (pH 7.2; HanLAB), 150 mM NaCl (Fisher Scientific), 0.1% Triton X-100 (Sigma-Aldrich), 5 mM ethylenediaminetetraacetic acid (EDTA; HanLAB), 1% sodium dodecyl sulfate (Bio-Rad Laboratories Inc.), 1 mM dithiothreitol (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (PMSF; MP Biomedicals), and 1X protease inhibitor (Roche). We used a Kinematica Polytron homogenizer (Kinematica AG), and centrifuged the samples at 15,928 × g for 20 minutes at 4 °C. Protein concentrations were determined by bicinchoninic acid protein assay (Thermo Fisher Scientific). Lysates (10 μg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose membranes (Bio-Rad). After Western blotting, the chemiluminescence signals were detected using a West Femto kit (SuperSignal, Thermo Fisher Scientific). The intensity of the signal was normalized against the β-tubulin signal. The data were presented as mean±standard error of the mean. The primary antibodies used were rabbit polyclonal anti-ATG9A antibody (26276-1-AP; Proteintech Genomics), rabbit polyclonal anti-ATG9B (ab117591; Abcam), and rabbit polyclonal anti-β-tubulin antibody (ab6046; Abcam).

Lee M., Son S., Lim H.J, & Song H. (2024). The differential expression patterns of Atg9a and Atg9b in cells of the reproductive organs. Clinical and experimental reproductive medicine.

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Isolation and Characterization of Cytoskeletal Proteins

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Ficoll-Paque was obtained from Pharmacia (Uppsala, Sweden). Fibronectin was from Calbiochem (La Jolla, San Diego, CA, USA). Bicarbonate-free Hank’s solution, Ca2⁺-free Dulbecco PBS, cytochalasin D, minoxidil, doxycycline, wortmannin, Akt 1/2 inhibitor, cytochalasin D, latrunculin A, blebbistatin, staurosporine, 4-bromophenacyl bromide, and E64 were obtained from Sigma (Steinheim, Germany). Analytical chromatography conditions: eluent MCI Buffer L-8800-PH-1–4 and ninhydrin coloring solution kit for Hitachi 29,970,501 (Wako Chemicals, North Chesterfield, VA, USA). Coomassie Brilliant Blue G-250 was obtained from Serva, PMSF from MP Biomedical, trypan blue from Fluka AG, trypsin from Promega, glutaraldehyde from Ted Pella, carboxy-H₂DCF-DA from Molecular Probe, (Eugene, OR, USA).

Galkina S.I., Fedorova N.V., Ksenofontov A.L., Serebryakova M.V., Golenkina E.A., Stadnichuk V.I., Baratova L.A, & Sud’ina G.F. (2021). Neutrophil Adhesion and the Release of the Free Amino Acid Hydroxylysine. Cells, 10(3), 563.

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Limonoid Extraction and Characterization

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MES, heparin, BES, BSA, BCA, CuSO₄, ThS and ANS were purchased from Sigma. IPTG and DTT were purchased from Calbiochem. Other chemicals such as ampicillin, NaCl, KCl, Na₂HPO₄, KH₂PO₄, EGTA, MgCl₂, PMSF, ammonium acetate were from MP and protease inhibitor cocktail was from Rosche. Dulbecco modified Eagle’s media (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin were purchased from Gibco (Thermo fisher Scientific), and MTT (Thiazolyl blue tetrazolium bromide) was purchased from Sigma. The basic limonoids used here were isolated from fruit of A. indica. These compounds were characterized and well matched with that of reported data³⁵.

Gorantla N.V., Das R., Chidambaram H., Dubey T., Mulani F.A., Thulasiram H.V, & Chinnathambi S. (2020). Basic Limonoid modulates Chaperone-mediated Proteostasis and dissolve Tau fibrils. Scientific Reports, 10, 4023.

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Alzheimer's Disease Protein Aggregation Study

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Baicalein, ThS, ANS, BES, and MTT were purchased from Sigma. Other reagents such as NaCl, Sodium azide, heparin, MgCl₂, EGTA and PMSF were obtained from MP Biomedicals. DTT was obtained from Calbiochem and protease inhibitor cocktail was purchased from Roche. pan-Tau K9JA was purchased from Dako (A-0024) and secondary goat anti-rabbit antibody conjugated to HRP was purchased from Thermo Fisher Scientific (A16110). Hybond-PVDF 10600087 membrane 0.45 μm was obtained from Amersham Biosciences. The neuroblastoma N2a (ATCC: CCL-131 Neuro-2a Neuroblastoma mouse) cells were purchased from ATCC. The cell culture reagents were purchased form Invitrogen. 400 carbon coated copper grids were purchased from Ted Pella (01822-F). 10 mM and 1 mM stock solutions of Baicalein were prepared in ethanol. 12.5 mM mother stock of ThS was prepared in 1:1 ethanol: miliQ water, which was further diluted to 200 μM stock in filtered miliQ water. ANS was prepared as 10 mM stock in filtered miliQ.

Sonawane S.K., Uversky V.N, & Chinnathambi S. (2021). Baicalein inhibits heparin-induced Tau aggregation by initializing non-toxic Tau oligomer formation. Cell Communication and Signaling : CCS, 19, 16.

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Neurosphere Lysis and Western Blot Analysis

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Neurosphere cells cultured with different growth factor combinations for 3 div were harvested by centrifugation and lysed in lysis buffer (50 mM Tris/Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% (v/v) Triton-X100, 0.1% (w/v) deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 40 mM sodium fluoride, 1 mM orthovanadate, pH 10) supplemented with the protease inhibitors PMSF (1 mM, MP Biomedicals), IAA (18.5 µg/ml, Sigma-Aldrich), SBTI (10 µg/ml, Sigma-Aldrich), Aprotinin (10 µg/ml, Sigma-Aldrich), Leupeptin (0.5 µg/ml, Sigma-Aldrich), and Pepstatin (0.1 µg/ml, Sigma-Aldrich) for 30 min on ice before the debris was removed by centrifugation. The conditioned medium was directly used for protein analysis. The lysates and supernatants were fractionated on a 4–10% gradient SDS polyacrylamide gel together with the Precision Plus Protein Dual Color Standard (Bio-Rad) and semi-dry blotted to methanol-activated polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated with antibodies for Tnc (polyclonal anti-Tnc, rabbit (batch KAF14), 1:3,000) and α-tubulin (DM1α, 1:10,000, Sigma-Aldrich), probed with appropriate HRP-coupled secondary antibodies (1:10,000, Dianova), and developed with the Clarity Western Blot ECL substrate (Bio-Rad).

Theocharidis U., Roll L, & Faissner A. (2021). The expression of tenascin-C in neural stem/progenitor cells is stimulated by the growth factors EGF and FGF-2, but not by TGFβ1. Cell and Tissue Research, 385(3), 659-674.

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Western Blot Analysis of KLF12 in A549 Cells

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Briefly, A549 cells were homogenized in ice‐cold RIPA buffer mixing with PMSF (MP Biomedicals, Solon, OH, USA). After 30 min of incubation on ice and brief sonication, protein concentrations were determined using BCA protein assay kit (Thermo Scientific, USA). The assay was performed as described previously,18 (link) with KLF12 primary antibody and GADPH primary antibody (as control). Immunoreactive proteins were measured using eZwest Lite Auto Western Blotting System (GenScript, Nanjing, China).

Zhao J., Yu H., Han T, & Zhu X. (2021). Prognosis Value of microRNA-3677-3p in Lung Adenocarcinoma and Its Regulatory Effect on Tumor Progression. Cancer Management and Research, 13, 9261-9270.

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Characterization of Pancreatic Cell Lineages

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Cells were harvested at DE and PP stages and lysed using cell lysis buffer (Cell Signaling Technology, #9803) containing protease inhibitor (Roche, #05892791001) and 1mM PMSF (MP Biomedicals, #ICN19538105). Samples preparation complied with NuPAGE® Novex protocol and samples were separated by 10% Bis-Tris Gel (Novex, #NP0301BOX). The protein samples were then transferred to Nitrocellulose Pre-Cut Blotting Membranes (Novex, #LC2001) followed by blocking with 5% milk in Tris-based saline with Tween 20 (0.1% TBST) buffer for 1 hour at R.T.. The membrane was incubated with primary antibodies overnight at 4°C, followed by incubation with HRP conjugated secondary antibodies at R.T. for 1 hour. ECL western blotting detection reagent (Amersham, #RPN2236) was used to visualize the protein bands. The following antibodies were used with the dilution ratio noted: rabbit anti-GATA6 (Cell Signaling Technology, #5851S, 1:1,000), mouse anti-GATA4 (BD, #560327, 1:100), goat anti-PDX1 (R&D, #AF2419, 1:1,000), mouse anti-ACTB (β-actin) (Cell Signaling Technology, #3700S, 1:10,000)

Shi Z.D., Lee K., Yang D., Amin S., Verma N., Li Q.V., Zhu Z., Soh C.L., Kumar R., Evans T., Chen S, & Huangfu D. (2017). Genome editing in hPSCs reveals GATA6 haploinsufficiency and a modifying genetic interaction with GATA4 in human pancreatic development. Cell stem cell, 20(5), 675-688.e6.

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