CaMKII activity was measured using the ‘SignaTECT Calcium/Calmodulin-Dependent Protein Kinase Assay System’ (Promega Corporation, Madison, WI). Mouse hearts were homogenized in a lysis buffer solution containing 20 mM Tris–HCl (pH 8.0), 2 mM EDTA, 2 mM EGTA, 2 mM DTT, PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and cOmplete, Mini protease inhibitor cocktail (Roche Diagnostics). Hearts were subjected to three 5 s pulses at 12,000–17,000 rpm using a PRO200 Homogenizer Unit (Pro Scientific, Oxford, CT). The homogenate was centrifuged at 2000 rpm for 10 min and the resultant supernatant was collected and assayed for CaMKII activity as per the manufacturers instructions. To determine the effect of MLCKp on CaMKII activity, 0.1 or 1 μM MLCKp was added to the reaction.
Mini protease inhibitor cocktail
Manufactured by Roche
Sourced in United States, Germany, Switzerland, United Kingdom
The Mini Protease Inhibitor Cocktail is a concentrated solution designed to inhibit a broad spectrum of serine, cysteine, and metalloproteases. It is suitable for use in the preparation of protein extracts from a variety of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Phosstop, by Roche (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Phosstop, by Merck Group (3 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions)
β actin, by Merck Group (2 mentions)
Immobilon pvdf membrane, by Merck Group (2 mentions)
Nupage 4 20 gel, by Thermo Fisher Scientific (2 mentions)
Nonidet p 40, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Bradford protein assay, by Bio-Rad (2 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
71 protocols using mini protease inhibitor cocktail
1
Measuring CaMKII Activity in Mouse Hearts
2
Murine Lung Lavage and Tissue Harvest
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After measurement on the flexiVent, mice were removed from ventilation, and quickly exsanguinated. Bronchoalveolar lavage (BAL) fluid was then collected by flushing the lungs with 1 mL of cold PBS with complete, Mini Protease Inhibitor Cocktail (Roche, San Luis Obispo, CA, United States). BAL was processed the same day, total cell count was conducted and slides were prepared using Cytospin. Organs were then dissected and flash frozen in liquid nitrogen and stored at −80°C until analysis.
3
Western Blot Analysis of Recombinant Proteins
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Thawed and washed cell pellets were lysed in 1 mL sterile PBS with 10% (v/v) glycerol and 1x cOmplete mini protease inhibitor cocktail (Roche). Ten micrograms of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were probed with α-FLAG M2 (Sigma-Aldrich F1804, 1:1,000 dilution) or α-GroEL (Santa Cruz #5177, 1:5,000), followed by IRDye 800CW Goat anti-Mouse IgG secondary antibody (LI-COR, 1:15,000) and imaged (Odyssey Cx scanner, LI-COR).
4
Protein Expression Analysis in HK-2 Cells
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Proteins were obtained from HK-2 cell lysates and animal tissue, using RIPA buffer (Thermo, USA) containing Complete, Mini Protease Inhibitor Cocktail (Roche, USA) and Halt Phosphatase Inhibitor Cocktail (Thermo, USA). To examine the expression of proteins, separated protein in SDS/PAGE (gradient gels) were transferred onto polyvinylidene difluoride membranes (PVDF) (Bio-rad, USA), and then incubated with 5% BSA (sigma) for blocking. The membranes were incubated with anti-GAPDH antibody (Santacruz, USA), anti-type 1 collagen antibody (abcam, UK), anti-fibronectin antibody (abcam), anti-α-SMA antibody, anti-Vimentin antibody, anti-Lin28a antibody, anti-phospho-ERK antibody, anti-ERK antibody, anti-phospho-JNK antibody, anti-JNK antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-phospho-smad3 antibody and anti-smad3 antibody (Cell signaling, USA). After incubating with HRP-linked antibody (Cell signaling), the protein expression on blots was detected by ChemiDocTMXRS+ (Bio-rad) and the bands were quantified using the Image LabTM Software (Bio-rad).
5
Protein Extraction and Western Blot Analysis
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Plant material (100 mg) treated or not with MG132, was homogenized and extracted in protein extraction buffer [50 mM Tris-HCl pH8, 150 mM NaCl, 10 mM EDTA, 50 mM NaFluoride, 1% NP40, 0.5% Deoxycholate, 0.1% SDS and protease inhibitors (cOmplete, Mini Protease Inhibitor Cocktail from Roche)]. Samples were cleared by centrifugation at 13,000 g for 20 min at 4°C and proteins extracted in 1X SDS-Laemmli buffer. Western blot was performed as described previously (Durut et al., 2014 (link)) using, α-H3 (CT, pan from Millipore) α-NUC1 (Pontvianne et al., 2010 (link)), α-RPN1a (Wang et al., 2009 (link)), α-RPN10 (Lin et al., 2011 (link)), and α-PRXII (Bréhélin et al., 2003 (link)) antibodies.
6
Chromatin Immunoprecipitation of RNA Polymerase
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The overnight bacteria cultures were transferred to a fresh medium containing the appropriate antibiotics until the mid-log-phase (OD600 = 0.6) was reached. The cross-link was performed by adding formaldehyde to a 1% concentration for 10 min and quenching with glycine. Then, the bacteria were pelleted and washed with a Tris buffer (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl). Prior to sonication, the bacteria were resuspended in in IP buffer (50 mM HEPES–KOH [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and mini-protease inhibitor cocktail [Roche]). The cell lysis was centrifuged, and the supernatant was incubated with RNA Polymerase ImmunoAffinity Resin (number 673601). The RNAP binding DNA fragments were eluted after proteinase K digestion. The purified DNA fragments were used to perform downstream qPCR.
7
Extraction and Fractionation of Cellular Proteins
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The cellular and cytoplasmic extract proteins were obtained as previously described (11 (link)). Briefly, cells were harvested by trypsinizing and the whole cell protein was acquired by lysing the cells on ice for 20 min in 700 μl lysis buffer with protease inhibitors and mini protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The lysate was then centrifuged at 12,000 × g for 20 min and the supernatant was collected, and stored at −80°C. To prepare the cytoplasmic proteins, the cell pellets were suspended in 500 μl lysis buffer (see whole cell instructions) without Tween-20 detergent, samples were sonicated (1 sec × 30) on ice and then centrifuged at 10,000 × g for 20 min. The supernatant (cytoplasmic fraction) was collected in accordance with the method described in our previous report (11 (link)).
8
Proteomic Analysis of P. aeruginosa
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P. aeruginosa PAO1 cells (OD600 = 0.5, 30 mL) were grown at 37°C in 40 mL of MOPS with succinate (30 mM) or propionate (40 mM) as the sole carbon source and good aeration (shaking at 250 rpm) in baffled flasks (500-mL volume). Cultures were grown and analyzed in triplicate. The cell pellets were resuspended in 2 mL of lysis buffer (100 mM Tris-HCl, 50 mM NaCl, 10% [vol/vol] glycerol, and 1 mM tris(2-carboxyethyl)phosphine [TCEP], pH 7.5) containing one cOmplete Mini protease inhibitor cocktail (Roche). Following three rounds of sonication (3 × 10 s) on ice, supernatants were clarified by sedimentation (21,130 × g, 15 min, 4°C) in an Eppendorf 5424R centrifuge. Aliquots (100 μg) of each sample were reduced with TCEP, alkylated with iodoacetamide, and labeled with tandem mass tags (TMTs). TMT labeling was carried out according to the manufacturer’s protocol.
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9
Immunization-Induced Lymph Node Cytokine Analysis
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To determine the cytokine/chemokine content of lymph nodes, animals were vaccinated and inguinal lymph nodes were collected 6–48 h post immunization. For IFNAR-1 blockade, an antagonistic mAb (clone: MAR1-5A3, BioXcell cat# BP0241) or an isotype control (clone: MOPC-21, BioXcell cat# BP0083) were injected intraperitoneally 24 h prior to immunization. Protein Extraction Buffer (Invitrogen, cat# EPX-9999-000) containing Mini protease inhibitor cocktail (Roche, cat# 53945000) and HALT phosphatase inhibitors (Thermo Fisher, cat# 78442) was added to the intact lymph nodes prior to homogenization using a TissueLyser II (Qiagen). Centrifugation-cleared lysates were analyzed with Luminex Cytokine and Chemokine kits (EMDMillipore, cat# MCYTOMAG-70K and MECY2MAG-73K).
10
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from human HCC cells by using a modified radioimmunoprecipitation assay buffer (150 mM NaCl, 1% NP240, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 7.4) in the presence of cOmplete, Mini protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Western blot analysis was performed after the separation of 50 μg of proteins on a sodium dodecyl sulfate (SDS)/4%–20% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) with conventional methods and an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ, USA). After that, filters were probed with mouse anti-hPTPRJ 1:100, hamster anti-mouse PTPRJ 1:1,000, and rabbit anti-GAPDH (Sigma-Aldrich Co., St Louis, MO, USA) 1:1,000 plus HRP-conjugated corresponding secondary antibodies at 1:1,000 (Cell Signalling Technologies, Danvers, MA, USA). Signals were visualized with SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific) by exposure to films.
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