Hearts were immersed in liquid nitrogen and grinded using porcelain mortar and pestle. A portion of 50 mg of the tissue powder was transferred to 1 mL of PureZol RNA isolation reagent (BioRad, Hercules, CA, USA) and homogenized by Pellet Pestle® Motor (Kimble Kontes, St. Louis, MO, USA). Samples were frozen at −80 °C until further processing. Another portion of 50 mg of tissue powder was mixed with homogenization buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Triton X-100, and Protease Inhibitors Cocktail Set III (Sigma-Aldrich, St. Louis, MO, USA) and homogenized on ice by Pellet Pestle® Motor (Kimble Kontes, St. Louis, MO, USA) to prepare 20% tissue homogenates (w:v). Homogenization buffer for preparation of samples for phospho-MYL9 Western blot determination additionally contained 1% of Phosphatase Inhibitor Cocktail 2 and 1% of Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich, St. Louis, MO, USA). Samples were centrifuged at 4 °C and the protein content in the supernatant was measured using Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) using Bovine Serum Albumin (BSA, heat shock fraction, Sigma-Aldrich, St. Louis, MO, USA) as a protein standard. Supernatants were stored at −80 °C until biochemical analysis.
Protease inhibitors cocktail set 3
Manufactured by Merck Group
Sourced in United States, Canada
Protease Inhibitors Cocktail Set III is a laboratory product offered by Merck Group. It is a mixture of compounds designed to inhibit the activity of proteases, which are enzymes that break down proteins. The set includes a selection of protease inhibitors that can be used in various research applications, such as protein extraction and purification.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Bio-Rad (3 mentions)
Bovine serum albumin heat shock fraction, by Merck Group (1 mentions)
Purezol rna isolation reagent, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Gs 800, by Bio-Rad (1 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
4 protocols using protease inhibitors cocktail set 3
1
Cardiac Tissue Homogenization and Protein Extraction
2
Cardiac Tissue Characterization Protocol
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The study was approved by the Animal Ethics Committee of the Polish Academy of Science (decision No. 25/2012). All steps of the experiments have been conducted in a way to avoid animal suffering. On day 21, after echocardiography was performed, all animals were subjected to anesthesia with ketamine (10 mg/kg body weight) and then sacrificed by decapitation. The hearts were collected and divided into two parts. One part of each heart was rapidly deep-frozen and crushed into a powder at liquid nitrogen temperature and stored at −80 °C for subsequent biochemical analysis. The second part of each heart was processed for transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining. Prior to zymography and western blotting a proper amount of heart powder was mixed with homogenization buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% Triton X-100, and Protease Inhibitors Cocktail Set III (Sigma-Aldrich) to prepare 20% homogenates (w/v) with subsequent mechanical homogenization (3 times for 10 s on ice) and centrifugation (10,000× g, for 5 min at 4 °C). Protein content in supernatants was measured with Bradford Protein Assay (Bio-Rad, Warszawa, Poland) and bovine serum albumin (heat shock fraction, ≥98%, Sigma-Aldrich) served as a protein standard.
3
Protein Extraction and Purification
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Sodium chloride (NaCl; FW 58.44, ACS reagent, ≥ 99%), hydrochloric acid (HCl; ACS reagent, 37%), sodium hydroxide (NaOH, 10.0 N), protease inhibitors cocktail (Set III, EDTA‐Free), and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Sigma‐Aldrich (Millipore Sigma, Canada). Human serum albumin (HSA, 100 μg.μL−1) and Dulbecco's phosphate‐buffered saline (10X PBS) were purchased from Invitrocare (Invitrocare Inc.) and Thermo Fisher (Thermo Fisher Scientific, Canada), respectively.
4
Quantification of Heart Tissue MMP-2 and MMP-9
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Prior to the biochemical analysis, the heart tissue powder was homogenized in the buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Triton X-100, and protease inhibitors Cocktail Set III (Sigma-Aldrich). In homogenates, protein content was analyzed using Bradford protein assay (Bio-Rad, Warszawa, Poland), and bovine serum albumin was used as a protein standard. The activity of MMP-2 and MMP-9 was assessed in heart extracts by gelatine zymography. Equal total protein samples were applied to 7.5% polyacrylamide gels copolymerized with gelatine (2 mg/mL), containing 0.1% sodium dodecyl sulfate (SDS). In following electrophoresis (100 V, 4 C), each gel was washed 3 times for 20 minutes in 2.5% Triton X-100 and then placed in incubation buffer (50 mmol/L Tris-HCl, 10 mmol/L CaCl 2 , 200 mmol/L NaCl, and 0.05% NaN 3 ) at 37 C for 18 hours. After incubation, each gel was stained in 0.5% Coomassie Brilliant Blue R-250 (Bio-rad), 30% methanol, and 10% acetic acid for 2 hours and then destained in 30% methanol/10% acetic acid until the bands were clearly visible. Gels were scanned using GS-800 (Bio-rad) calibrated densitometer with Quantity One v4.6.9 software (Bio-Rad), and the relative MMPs activity was determined and expressed in arbitrary units calculated on the basis of recombined MMP standard activity.
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