Edta free protease inhibitor cocktail tablet

Kidney cortical tissue was prepared for immunoblot analysis with antibodies against phosphorylated and total Drp1. Kidney cortex samples were homogenized in extraction buffer containing 50 mM HEPES, pH7.4, 150 mM NaCl, 0.5% Triton X-100, 0.025 mM ZnCl2, 0.1 mM Pefabloc SC Plus (Roche, Basel, Switzerland), EDTA-free protease inhibitor cocktail tablet (Roche, Basel, Switzerland), and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined using the Micro BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).
Western blot was performed by separating 15 µg of total protein in 7% SDS-polyacrylamide gels and transferring it onto PVDF membranes (Immobilon-P Millipore, Burlington, MA, USA). Membranes were incubated in skimmed milk blocking solution (5%) for 1 h and incubated overnight at 4 °C with galectin-3 (1:500; 126701, Biolegend, San Diego, CA, USA) in 2.5% skimmed milk. HRP-conjugated anti-mouse IgG antibody was used as secondary antibody. β-Actin antibody (1:20,000; BS1003, Bioworld, Dublin, Ireland) was used as loading control.
Proteins were detected in films (AGFA CURIX, Mortsel, Belgium) after 3-min incubation with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Protein bands were quantified by densitometry with the ImageJ software.

Neutrophils (5 × 10⁶/mL) in HEPES buffered RPMI 1640 supplemented with 0.5% (w/v) HSA were incubated for 15 min at 37 °C. Subsequently, neutrophils of COPD patients and healthy age-matched controls were immediately prepared for protein extracts. The neutrophils (1 × 10⁷/sample) were washed twice with sucrose buffer (0.34 M sucrose, 1 mM EDTA, 10 mM Tris) and lysed in lysis buffer (10 mM Tris pH 7.4, 10% glycerol, 1% NP40, 50 mM NaF, 20 mM tetra-Na pyrophosphate, 1 mM DTT, 2 mM vanadate, 1 mM PMSF, 2 mM DFP and 1 × Complete EDTA-free protease inhibitor cocktail tablet (Roche)). Proteins were precipitated with 80% acetone and dissolved in labeling buffer (8 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM Tris pH 8.5).

Frozen cells were thawed and re-suspended in 15 mL of a lysis buffer containing 1 M NaCl, 50 mM HEPES (pH 7.6), 10 mM imidazole, 1 mM MgSO4, 18 mM B-mercaptoethanol, 1 mL B-PER (Thermo Scientific), and an EDTA-free protease inhibitor cocktail tablet (Roche). Cells were lysed by sonication (3 cycles × 2 min/cycle) on ice. The lysate was centrifuged at 28,000× g for 30 min at 4 °C. The supernatant was syringe-filtered to produce a clear lysate. The lysate was loaded onto an IMAC column packed with Ni⁺²-charged NTA-resin (Amersham). Protein was then eluted from the column with a buffer consisting of 0.5 M NaCl, 50 mM HEPES (pH 7.6), and a stepped gradient of imidazole of 10, 25, and 300 mM, where the protein eluted in the 300 mM imidazole fraction. The protein was further purified using a Superdex 26/60 S75 preparative-grade gel filtration column (GE Amersham) with a buffer consisting of 150 mM NaCl, 25 mM HEPES (pH 7.4), 1 mM DTT, and 0.25 mM sodium azide.

Cell pellets were washed once with 50 mM Tris HCl, pH 7.5, and 0.5 M NaCl and resuspended (20% w/v) in 50 mM Tris HCl, pH 7.5, 0.5 M NaCl, 30 μg/ml DNase (Worthington), one EDTA-free protease inhibitor cocktail tablet (Roche), 1 mM CaCl₂, and 1 mM MgCl₂ and ruptured first by tip-sonication and then using an EmulsiFlex-C3 homogenizer (Avestin) at 12,000 psi external pressure. Debris and unbroken cells were removed by centrifugation (10,000g for 20 min), and the membranes were collected by ultracentrifugation at 135,000g for 30 min, washed once in 25 mM Tris HCl, pH 7.5, 0.1 M NaCl, and resuspended in the same buffer to ∼1 mg/ml.

The cultured supernatants and cell lysates were collected at the indicated time points after Stx2 treatment, and protein in the cell-free supernatants was concentrated using the methanol-chloroform precipitation method (27 (link)). The cell pellets were lysed with the RIPA Lysis buffer (89901, Thermo) supplemented with a 1:50 diluted protease inhibitor cocktail tablet (EDTA-free protease inhibitor cocktail tablet, Roche). Such prepared samples were then mixed with the equal volume of 2× SDS-loading buffer and detected for pro-IL-1β/IL-1β and pro-caspase-1/caspase-1 by immunoblotting. β-Actin was used as the positive control. Immobilized proteins were incubated with primary antibodies against IL-1β (sc-52012; 1:1,000), caspase-1 (AG-20B-0042; 1:1,000), and β-actin (4967S; 1:1,000), and followed by incubation with the secondary antibodies (IRDye 800-labeled anti-rabbit IgG; 611-132-002; 1:10,000 (Santa Cruz Biotechnology). The protein levels were detected using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).

After removal of the biliary brush from the working channel of the endoscope the brush was advanced out of the sheath, cut and placed into a storage buffer (Varleigh Dx (UK) Ltd, London, UK), which contained one complete mini EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics Ltd, Lewes, East Sussex, UK) per 10 ml of buffer. The sample was gently agitated before being rapidly frozen to −80 °C within 4 h of the procedure.

Sarkosyl-insoluble tau was isolated from the brainstem of 5- to 6-month-old rats. Frozen brain samples of the studied rats were homogenised in 10 vol of ice-cold extraction buffer (SL buffer: 20 mM Tris, pH 7.4; 800 mM NaCl; 1 mM ethylene glycol tetraacetic acid), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% β-mercaptoethanol, 10% sucrose, 1 mM Na₃VO₄, 20 mM NaF, supplemented with EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA) using an OMNI TH tissue homogeniser (OMNI International, Kennesaw, GA, USA). After 5-minute incubation on ice, the homogenates were cleared by centrifugation at 20,000 × g for 20 minutes at 4 °C. Solid sarkosyl (N-lauroyl sarcosine, Na-salt; Sigma-Aldrich) was added to the supernatant to achieve 1% concentration and stirred for 1 h. Thereafter it was centrifuged at 100,000 × g for 1.5 h at room temperature. The supernatant was collected, and pellets were gently rinsed with 1 ml of the SL buffer and centrifuged for 20 minutes at room temperature. The pellets were dissolved in sodium dodecyl sulphate (SDS) sample loading buffer.