The PC and the MOB were dissected and snap frozen for further analyses. The tissue was homogenized in RIPA lysis buffer containing protease inhibitory cocktail (Sigma-Aldrich) on ice for 30 min. Total protein concentration was determined by Lowry assay (Bio-Rad Laboratories, US). Samples containing 4 μg/μl total protein were mixed with LDS sample buffer (Novex, Life Technologies, US) and incubated in 95 °C for 5 min. After electrophoresis on Bolt 4–12% Bis-TrisPlus gel (ThermoFisher Scientific, US), proteins were transferred on nitrocellulose membrane (iBlot 2 Dry Blotting System, 25 V for 6 min, ThermoFisher, US). Immuno-detection was performed with antibodies against: 1) PV (1:500, polyclonal, rabbit, Abcam, US), 2) CB (1:100 polyclonal, rabbit antibody, Abcam), 3) CR (1:1000, monoclonal, rabbit, Abcam, US), and 4) β-actin (1:450, monoclonal, mouse, SantaCruz Biotechnology). Secondary antibodies used: bovine anti-rabbit (1:10,000; SantaCruz Biotechnology) and goat anti-mouse (1:5000; SantaCruz Biotechnology, US) were conjugated with horseradish peroxidase (HRP). Immuno-reactive bands were developed using ECL substrate for HRP (GE Healthcare, Sweden) and documented with a GelDoc chemiluminescent system (Bio-Rad Laboratories, US). Normalization against β-actin was performed.
Protease inhibitory cocktail
Manufactured by Merck Group
Sourced in United States
The Protease Inhibitory Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. The cocktail contains a mixture of specific chemical compounds that target and inactivate various types of proteases, helping to preserve the integrity of protein samples during analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Lowry assay, by Bio-Rad (1 mentions)
Bovine anti rabbit, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Magpix system, by Merck Group (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
Trypsin gold, by Promega (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Tmtsixplex isobaric label reagent, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Mk 801, by Merck Group (1 mentions)
Evans blue, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by ITW Reagents (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
11 protocols using protease inhibitory cocktail
1
Western Blot Analysis of Neuronal Markers
2
Cytokine Profiling in Mouse Nerves
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Sciatic nerves and DRGs were rapidly harvested from mice perfused intracardially with cold saline, snap‐frozen in liquid nitrogen and kept at −80°C. The two sciatic nerves for each mouse were pooled for protein tissue extraction. Similarly, all the DRGs from lumbar to cervical segments were pooled for protein tissue extraction. For tissue processing, sciatic nerves and DRGs were manually homogenized in HEPES buffer (HEPES 6.25 mmol/L, IGEPAL 2%, PMSF 1 mmol/L, EGTA 1 mmol/L (pH 8), EDTA 16.9 mmol/L (pH 8), MgCl2 5 mmol/L) adding 10 μL/mL of protease inhibitory cocktail (Sigma) and PhosSTOP phosphatase inhibitor cocktail (Roche). Protein concentration was set to 3.3 μg/μL in DRG samples and 3 μg/μL in sciatic nerve samples. The protein levels of different cytokines were then determined and analyzed using a custom‐designed Milliplex Cytokine/Chemokine Magnetic Bead Panel on a MAGPIX system (EMD Millipore) in accordance with the manufacturer's protocol. Standard curves were generated using the specific standards supplied by the manufacturer. Data were normalized by the total protein concentration and afterward by the mean of the control samples.
3
Isolation of Mammary ECM Extracellular Matrix
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Extracellular matrix (ECM) was isolated from mammary gland using a homogenization method (O’Brien et al., 2010). Briefly, frozen mammary gland tissue was pulverized into fine powder using a mortar and pestle containing liquid nitrogen. Homogenization of powdered tissue (1 g) was performed in high salt buffer (2 ml) composed of 200 μg/ml phenyl methyl sulfonyl fluoride (PMSF), 2 mM n-ethylmaleimide (NEM) and protease inhibitory cocktail (Sigma). ECM was dialysed sequentially to eliminate urea and protease inhibitors. Ham’s F12 and M199 (1:1 ratio) media without serum was used for the final dialysis of ECM and ECM was removed from the dialysis membrane, aliquoted and stored at -20°C until use.
4
Circadian Clock Protein Expression
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Thoracic aorta and HUVEC cells were homogenized using ice cold 1X RIPA buffer with protease inhibitory cocktail (Sigma Aldrich, USA). Total protein was quantified by Bradford assay. SDS PAGE (10% polyacrylamide gel) was performed with equal amount of protein loading, that were transferred to PVDF membrane (Bio-Rad, USA). Primary antibodies for Sirt1, Clock, BMAL1 (1:1000) were added followed by secondary anti-rabbit horseradish peroxidase antibody (1:1500)/ anti-mouse horseradish peroxidase antibody (1:5000). Blots were stripped with stripping buffer and re-probed with housekeeping beta-actin antibody (1:1000). Blots were developed using ECL reagent (Bio-Rad, CA, USA). Development was done on X-ray films.
5
Quantitative Proteomic Profiling with TMT
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For proteomic experiments, protein lysates were obtained using RIPA buffer (Thermo Fisher Scientific, UK) supplemented with protease inhibitory cocktail (Sigma-Aldrich, Gillingham, UK) and 0.3 mM Na3VO4. Protein concentration was determined with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, UK).
Samples were diluted to 1 mg/ml in 0.1 M TEAB, reduced with 10 mM DTT at 60°C, and alkylated with 25 mM IAA. Trypsinization was carried out for 16 hours at 37°C using Trypsin-Gold (Promega, Southampton, UK). Peptides were desalted using the Sep-Pak C18 cartridges (Waters, Elmstree, UK) before peptide labelling with the TMTsixplex™ Isobaric Label Reagent (Thermo Fisher Scientific, UK) kit according to the manufacturer’s instructions. Additional information can be found in theSupplementary Materials and Methods .
Samples were diluted to 1 mg/ml in 0.1 M TEAB, reduced with 10 mM DTT at 60°C, and alkylated with 25 mM IAA. Trypsinization was carried out for 16 hours at 37°C using Trypsin-Gold (Promega, Southampton, UK). Peptides were desalted using the Sep-Pak C18 cartridges (Waters, Elmstree, UK) before peptide labelling with the TMTsixplex™ Isobaric Label Reagent (Thermo Fisher Scientific, UK) kit according to the manufacturer’s instructions. Additional information can be found in the
6
Thioacetamide-Induced Liver Injury Model
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The following agents were used: thioacetamide (TAA, Titrachem, Bazar Kimia, Tehran, Iran), berberine (Ber, Sigma-Aldrich Co., USA), MK-801 (Sigma-Aldrich Co., USA), DMSO (Panreac, Milan, Italy), protease inhibitory cocktail (Sigma-Aldrich Co., USA), sodium thiopental (Nesdonal, Daroopakhsh Co., Tehran, Iran), ELISA Kits for oxidative stress and interleukins assay (ZellBio GmbH, Lonsee, Germany), Evans blue (Eb, Sigma-Aldrich Co., USA).
7
Two-dimensional Proteomic Analysis of Cell Lines
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Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco (Waltham, MA, USA), and fetal bovine serum (FBS) was also purchased from Gibco. Urea, CHAPS zwitterionic detergent (CHAPS), dithiothreitol (DTT), immobilized pH gradient (IPG) buffer, Tris-HCl, and IPG strips (17 cm, pH 4–7, linear) were purchased from Bio-Rad (Hercules, CA, USA). Collagenase (Type I), protease inhibitory cocktail, and thioUrea were purchased from Sigma-Aldrich (St Louis, MO, USA). Sodium dodecyl sulfate (SDS), glycerol, Tris base, agarose, glycine, and Coomassie R-250 were purchased from Biosharp (Hefei, China). Iodoacetamide was purchased from TCI (Portland, OR, USA). Bromophenol blue was purchased from Fisher Scientific (Pittsburgh, PA, USA). TRIzol reagent and reverse transcription kit were purchased from Takara (Shiga, Japan). Fast SYBR Green Master Mix was purchased from Toyobo (Osaka, Japan).
8
Affinity Purification of Rif2-Interacting Proteins
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Sf9 cell lysate expressing MBP-Rif2-his was bound to amylose resin (NEB) and washed with wash buffer 1 M (50 mM Tris-HCl pH 7.5, 1 M NaCl, 0.2% [v/v] NP40, 2 mM EDTA, 1:1000 protease inhibitory cocktail (Sigma P8340) and then with wash buffer 0.1 M (same as wash buffer 1 M, but only 0.1 M NaCl). The recombinant MR variants (1 μg) were then added, incubated for 1 h at 4°C and the resin was then washed with wash buffer 0.1 M. As a negative control, the MR proteins were added to the amylose resin without MBP-Rif2-his to test for non-specific binding. The proteins were eluted with 0.1 M wash buffer supplemented with 20 mM maltose, and cleaved with PreScission protease. The eluates were analyzed by Ponceau staining or by western blotting with anti-His (MBL, D291-3, 1:5,000) or anti-Rad50 antibodies (Thermo Scientific, PA5-32176, 1:1,000).
9
Ventrolateral OFC Protein Extraction
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Mice were rapidly decapitated at P82; brains were frozen at –80°C and then sectioned into 1-mm sections. The ventrolateral OFC was dissected by a single experimenter using a 1-mm tissue core. Tissue was homogenized by sonication in lysis buffer [100 μl: 137 mM NaCl, 20 mM tris-Hcl (pH 8), 1% igepal, 10% glycerol, 1:100 Phosphatase Inhibitor Cocktails 2 and 3 (Sigma), 1:1000 Protease Inhibitory Cocktail (Sigma)]. Protein concentrations were determined by Bradford colorimetric assay (Pierce), and 15 μg/sample was separated by SDS-PAGE on a 12% gradient tris-glycine gel (Bio-Rad). Following transfer to PVDF membrane, membranes were blocked with 5% nonfat milk.
Primary antibodies were anti-PSD95 (Cell Signaling; 1:1000), with anti-HSP-70 (Santa Cruz; 1:5000) serving as a loading control. Immunoreactivity was assessed using a chemiluminescence substrate (Pierce) and measured using a ChemiDoc MP Imaging System (Bio-Rad).
Primary antibodies were anti-PSD95 (Cell Signaling; 1:1000), with anti-HSP-70 (Santa Cruz; 1:5000) serving as a loading control. Immunoreactivity was assessed using a chemiluminescence substrate (Pierce) and measured using a ChemiDoc MP Imaging System (Bio-Rad).
10
Analyzing Cellular Responses via Western Blot
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IMR90 cells were harvested following washing with PBS. Cells were lysed in a buffer containing 0.02M HEPES (pH 7.9), 0.4M NaCl, 0.1% NP-40, 10% (v/v) glycerol, 1 mM NaF, 1 mM sodium orthovanadate and a protease inhibitory cocktail (Sigma Chemical Co. St. Louis, MO). Extracts were subjected to Sodium Dodecyl Sulfate (SDS)-polyacrylamide gel electrophoresis, followed by blotting to PVDF. The blots were probed with antibodies from Santa Cruz to human CDCA2, ID4, p53, p21, β-actin and α-tubulin; from Cell Signaling to human phospho-ATM (Ser 1981) and phospho-p53 (Ser 15) ; from Sigma-Aldrich to human ATM.
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