The viability of the hPCISs was evaluated by intracellular ATP and protein concentration analyses as previously described (Li et al., 2017a (link); Martinec et al., 2019 (link)). ATP concentrations were measured, using a CLS II ATP bioluminescence assay kit (Roche, Mannheim, Germany), in hPCISs immediately following preparation and after both 24 and 48 h incubation, as well as in RIF-treated samples. ATP concentrations determined in hPCISs were normalized with respect to the protein content of pellets obtained during the process. For this purpose, they were dried overnight at 37°C and then solubilized in 200 μl of 5 M NaOH for 1 h at 37°C. Finally, Milli-Q water (800 μl) was added to the samples to give NaOH concentration of 1 M, and their protein contents were determined using a Lowry Protein Assay Kit (Bio-Rad DC Protein Assay, Veenendaal, Netherlands). The tissue with the ATP content above 1.5 nmol/mg of protein was considered viable (van de Kerkhof et al., 2008 (link)).
Lowry protein assay kit
Manufactured by Bio-Rad
Sourced in United States
The Lowry protein assay kit is a colorimetric method for the determination of protein concentration in solution. It is based on the Lowry reaction, which involves the reduction of the Folin-Ciocalteu reagent by protein in an alkaline medium. The resulting colored complex is measured spectrophotometrically.
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Lab products found in correlation
Enhanced chemiluminescence kit, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Western lightning chemiluminescence reagent plus, by PerkinElmer (2 mentions)
Atp bioluminescence assay kit cls 2, by Roche (1 mentions)
Ab1218, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Thermo scientific multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Mmp zymography assay kit, by Applygen (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Polyscreen pvdf membrane, by PerkinElmer (1 mentions)
Anti s6, by Cell Signaling Technology (1 mentions)
Anti ps6, by Cell Signaling Technology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
14 protocols using lowry protein assay kit
1
Evaluating hPCIS Viability via ATP and Protein
2
Immunoblotting of Fluorescent Fusion Proteins
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HEK293T cells were resuspended in lysis buffer and sonicated. After centrifugation (15,000 x g, 4 °C, 5 min), the protein concentration was determined in the lysate by a Lowry protein assay kit (Bio-Rad Laboratories GmbH, Munich, Germany). For immune detection of Clover, mRuby2, Clover fused (63bp) mRuby2, Clover-PPARγ1, mRuby2-RXRα, mRuby2-RXRα Δ414-462 and N-CoR2-mRuby2 constructs, 100 µg protein per sample was separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels followed by transfer onto nitrocellulose membranes (both Bio-Rad Laboratories GmbH) basically following standard procedures. Subsequently, membranes were incubated with first antibodies against GFP (1:1,000; ab1218, Abcam plc, Cambridge, UK), human PPARγ (1:1,000; D69, Cell Signaling Technology, Danvers, USA), human RXRα (1:1,000; NB100-1466, Novus Biologicals, LLC, Littleton, USA), human N-CoR2 (1:1,000; ab2781, Abcam plc) and tRFP (1:1,000; AB233, Evrogen Joint Stock Company, Moscow, Russia) followed by Alexa Fluor® 488, 546 or 647 (Life Technologies Inc., Carlsbad, USA) fluorescent dyes secondary antibodies (1:10,000). Blots were visualized using the ChemiDoc XRS+ system (Bio-Rad Laboratories GmbH).
3
Quantification of Intestinal Cytokines in Rats
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Samples were prepared according to the manufacturer’s instructions, and the chemokines or cytokines were quantified using an ELISA kit. Concentrations of IL-6, IL-1β, IL-10, GM-CSF, IFN-γ, and TNF-α in the small intestines of the drug-treated rats (50 mg/kg of ACE) were determined using an ELISA kit (Ab frontier, South Korea) following the manufacturer’s protocol. The concentration of whole homogeneous lysates obtained from each sample was normalized and the lysates were diluted within the measurable range. Optical density was measured at 450 nm (A450) using a microplate reader (Thermo Scientific Multiskan GO, Microplate Spectrophotometer; Thermo Fisher Scientific). Briefly, small intestine tissue (100 mg) was washed with 1X PBS, and then 1 ml of 1X PBS was added to completely homogenize the isolated small intestine by using a tissue disperser homogenizer. Homogenates were centrifuged at 5,000 g for 5 min at 2–8°C. The supernatant was used to measure the enzyme activity. To normalize the protein levels in all supernatant samples, the protein concentration was quantified in each sample by using the Lowry protein assay kit (Bio-Rad, Hercules, CA, USA).
4
Quantifying Aortic MMP-2 and MMP-9 Activities
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Evaluating the presence of both activated and pro-forms of MMP-2 and MMP-9 has been described previously [28 (link)]. The MMP zymography assay kit was purchased from Applygen Technologies, Beijing, China. Briefly, frozen abdominal aortas were weighed and homogenized. The total protein was normalized using a Lowry protein assay kit (Bio-Rad) and resolved by 11% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) copolymerized with gelatin (1 mg/mL). Gels were washed in 2.5% Triton X-100 to remove SDS and allow renaturation of MMPs, transferred to developing buffer (50 mM Tris pH 7.5, 5 mM CaCl2, 1 μM ZnCl2), and incubated overnight at 37°C under continuous shaking. The activities of MMP-2 and MMP-9 were detected with a ChemiDoc MP Imaging system (Bio-Rad). The relative expression levels of proteins were normalized against the normal group.
5
Western Blot Analysis of Retinal Proteins
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Frozen retinas were lysed by sonication in lysis buffer (20 mm Na2HPO4, 250 mm NaCl, 30 mm NaPPi, 0.1% NP-40, 5 mm EDTA, 5 mm DTT) supplemented with protease inhibitor cocktail (Sigma-Aldrich). Lysates concentration was determined using a Lowry protein assay kit (BIO-RAD) following sonication and centrifugation. Protein supernatants were separated under denaturing conditions by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with antibodies (Table 1 ), as described (Roger et al., 2010 (link)). Proteins were visualized using enhanced chemiluminescence kit (BIO-RAD). α-Tubulin was used for normalization. Quantification was performed using ImageJ software (http://imagej.nih.gov/ij/ ; provided in the public domain by NIH).
6
Western Blot of Islet Proteins
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Freshly isolated or cultured islets were lysed in triple detergent lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, 0.1% SDS, 1% IGEPAL-CA630, 0.5% Sodium Deoxycholate, protease and phosphatase inhibitors), followed by sonication and frozen-thaw cycles. Protein concentration was measured with the Lowry protein assay kit (Bio-Rad, Hercules, CA, USA). Protein extracts (15–30 μg per replicate) were separated by SDS-PAGE electrophoresis onto 7.5–10% Tris-tricine gels and transferred to a Polyscreen PVDF membrane (Perkin Elmer, Waltham, MA, USA). Membranes were blocked for 1 h with TBS-0.05%Tween-5% BSA solution, followed by overnight incubation at 4 °C with the corresponding primary antibodies diluted in TBS.0.05%Tween: rabbit anti-MafA (1:250, Novus Biologicals), rabbit anti-p-Creb (S133), anti-Creb, anti-p-Erk1/2 (T202,Y204), anti-Erk, anti-p-Akt (T308), anti-Akt, anti-p-S6 (S235,S236), anti-S6 (1:1000, Cell Signaling Technology), and mouse anti-α-tubulin (1:1000; Sigma–Aldrich) as a loading control. Blots were visualized with ECL Reagent (Pierce Biotechnology, Rockford, USA) using a LAS4000 Lumi-Imager (Fuji Photo Fil, Valhalla, NY). Protein spots were quantified with Image Studio Lite V5.2 software.
7
Tumor Lysate Preparation Protocol
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Within 24 h following RFA, the ablation tissue was removed under sterile conditions. The tissue was added to a petri dish containing phosphate-buffered saline (PBS) prior to being sliced and ground until the tissue was a homogeneous slurry liquid. The resulting liquid was frozen in liquid nitrogen for 10 min and then thawed in a 37° water bath for 10 min. The freeze-thaw cycle was repeated five times in rapid succession, and the liquid was then centrifuged at 9,600 × g for 2 min. Finally, the supernatant was collected and sterilized through a 0.22 μm filter membrane. The protein content of the tumor lysate was determined using a Lowry protein assay kit, according to the manufacturer’s instructions (Bio-Rad). The samples were aliquoted and stored in liquid nitrogen until use.
8
Western Blot Analysis of Protein Abundance
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Total protein was extracted from aortas or HUVECs using radioimmunoprecipitation buffer (JRDUN Biotechnology Co., Ltd. Shanghai, China). The total protein concentration in each sample was measured using a Lowry protein assay kit (Bio Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of extracted protein (50 μg) were separated by SDS-PAGE on a 10% gel and transferred onto polyvinylidene difluoride membranes (Roche Diagnostics GmbH, Mannheim, Germany), followed by blocking in 5% skimmed milk overnight at 4°C. The protein abundance was detected with antibodies against HSP22 (1:1,000 dilution; cat. no. ab151552; Abcam), p-eNOS [1:500 dilution; cat. no. YS-(kt)-0446; YS Biotechnology Co., Ltd., Shanghai, China], eNOS (1:1,000 dilution; cat. no. ab76198; Abcam), p-p38 (1:1,000 dilution; cat. no. 4511), p38 (1:1,000 dilution; cat. no. 9212), anti-GAPDH (1:1,500; cat. no. 5174) and anti-β-actin (1:1,000; cat. no. 4970) (all Cell Signaling Technology, Inc.) for 2 h at room temperature. Next, the membranes were incubated with the aforementioned fluorescence secondary antibodies for 1 h at 37°C. Chemiluminescence detection was conducted using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Inc., Waltham, MA, USA). and signal intensity was determined using ImageJ software version 1.46 (National Institutes of Health, Bethesda, MD, USA).
9
Signaling Pathway Activation in ECFCs
10
Islet Protein Extraction and Western Blot
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Protein extraction of isolated islets was obtained using RIPA lysis buffer (Tris 50 mmol/l, pH 7.5, EDTA 5 mmol/l, NaCl, 1% 150 mmol/l, Triton X-100 1%, SDS 0.1%, 10 mmol/l sodium fluoride, 1% sodium deoxycholate, and protease inhibitors). For the extraction, islet lysates were frozen and thawed twice after centrifugation for 20 min at 4 °C, and supernatants were collected and stored at −80 °C. Protein quantification was determined with the Lowry protein assay kit (Bio-rad, Hercules, CA, USA). The protein was electrotransferred onto a PVDF membrane. The membranes were blocked for 1 h with 0.05% Tween-20 and 5% NFDM, and then incubated overnight at 4 °C with the antibodies: AntiPer1 (1:500; Thermo Scientific Inc.) ß-Actin was used as a loading control (1:5,000; GE Healthcare, Hertfordshire, UK). Protein bands were revealed by using the Pierce ECL western blot substrate (Thermo Fisher Scientific, Madrid, Spain). Respective bands were quantified by densitometry. Image J software 1.50a and intensity values for PER 1 were normalized with ß-Actin.
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