Chondrocytes were washed with PBS three times and lysed with RIPA supplemented with 1 mM protease inhibitor cocktail and 1 mM phosphatase inhibitor cocktail (Boster, Wuhan, China). Twenty five micrograms protein samples were separated on SDS-polyacrylamide gels and transfered onto a PVDF membrane. The membrane was firstly blocked with 5% bovine serum albumin (BSA) for 1 h and then incubated overnight with primary antibodies at 4°C. Subsequently, blots were washed with Tris-buffered saline with 0.1% Tween-20 (TBST) three times and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Finally, the immunoreactive bands were detected with the Western ECL System (Boster, Wuhan, China). GAPDH was used as the loading control and representative bands were shown. Western blots were repeated at least three times.
Protease inhibitor cocktail
Manufactured by Boster Bio
Sourced in China
The Protease Inhibitor Cocktail is a laboratory reagent designed to inhibit the activity of various proteases. It is a mixture of small-molecule compounds that target and inactivate specific proteolytic enzymes, preventing protein degradation in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by Boster Bio (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Ripa buffer, by Boster Bio (2 mentions)
Western ecl system, by Boster Bio (1 mentions)
Membrane protein extraction kit, by Beyotime (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Abca1, by Abcam (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
Bca assay, by Boster Bio (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Boster Bio (1 mentions)
Enhanced chemiluminescence, by Boster Bio (1 mentions)
Image labtm software, by Bio-Rad (1 mentions)
Protein a sepharose 4b, by Thermo Fisher Scientific (1 mentions)
Ab3443, by Abcam (1 mentions)
Anti hnf 1α, by Cell Signaling Technology (1 mentions)
Dpp 4 assay kit, by Enzo Life Sciences (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
15 protocols using protease inhibitor cocktail
1
Chondrocyte Protein Extraction and Western Blot
2
Membrane Protein Extraction and Western Blot
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The treated cells were washed with PBS and lysed in a RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Boster, Wuhan, China) to extract total proteins. The membrane protein was extracted from the cells using a Membrane Protein Extraction Kit (Beyotime, Shanghai, China). Immunoblotting analysis was performed as previously described (Yin et al., 2016 (link)) following standard procedures and using the primary antibodies, ABCA1, LDLR (Abcam), LXR (ABclonal), and Anti-β-actin (ProteinTech). In brief, the protein was separated on an 8–10% SDS denatured polyacrylamide gel and then transferred onto a NC membrane. The membranes were blocked with 5% skim milk and were incubated with appropriate antibodies at 4°C overnight. Then, the membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sanjian, Tianjin, China). The protein of interest was visualized using an Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific).
3
Quantitative Western Blotting Procedure
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Western blotting was performed as previously described (Dong et al., 2016 (link), 2017 (link)). Concisely, cells were treated with the RIPA Lysis Buffer (Boster) containing protease inhibitor cocktail (Boster). The concentration of total cell proteins was measured by the BCA assay (Boster). Equivalent quality of proteins was added in 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA, United States). After blocking with 5% bovine serum albumin, membranes were incubated overnight with corresponding antibodies at 4°C. Subsequently, the blot was washed and incubated with horseradish peroxidase -conjugated secondary antibodies (Boster) for 1h at room temperature. The immunoreactive proteins were determinated with enhanced chemiluminescence (Boster) and images were obtained by ChemiDocTM XRS + System with Image LabTM Software (Bio-Rad Laboratories).
4
Immunoprecipitation and Chromatin Immunoprecipitation Assays
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Immunoprecipitation assays were performed as described previously22 (link),23 (link) with slight modification. For ChIP assays, cross-linked chromatin was immunoprecipitated with 5 μg of antibody (anti-HNF-1α, Cell Signaling Technology, #89670; anti-PER1, Abcam, ab3443), or negative control rabbit IgG (Beyotime, A7016) at 4 °C overnight. Immunoprecipitated DNA was then used as a template for PCR. All primer sequences used for ChIP-PCRs were listed in Table S1 .
For co-immunoprecipitations, liver tissues were homogenized and lysed with a Non-denaturing lysis buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), and 1% NP-40 with protease inhibitor cocktail (Boster Biological Technology). To prepare immunoprecipitates, we incubated lysates with antibody (anti-HNF-1α, Cell Signaling Technology, #89670; anti-PER1, Abcam, ab3443) overnight at 4 °C, and then incubated with Protein A-Sepharose 4B (Invitrogen). Immunoprecipitates were washed five times with wash buffer containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.2 mM sodium orthovanadate with protease inhibitor cocktail, boiled in SDS–PAGE loading buffer. Proteins were analyzed by western blotting as described above.
For co-immunoprecipitations, liver tissues were homogenized and lysed with a Non-denaturing lysis buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), and 1% NP-40 with protease inhibitor cocktail (Boster Biological Technology). To prepare immunoprecipitates, we incubated lysates with antibody (anti-HNF-1α, Cell Signaling Technology, #89670; anti-PER1, Abcam, ab3443) overnight at 4 °C, and then incubated with Protein A-Sepharose 4B (Invitrogen). Immunoprecipitates were washed five times with wash buffer containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.2 mM sodium orthovanadate with protease inhibitor cocktail, boiled in SDS–PAGE loading buffer. Proteins were analyzed by western blotting as described above.
5
Quantitative Assessment of DPP-4 Activity
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To measure the activity of DPP-4, we used a DPP-4 Assay Kit (Enzo Life Sciences, Farmingdale, NY, USA) with the Gly-Pro-para-nitroaniline (pNA) chromogenic substrate, according to the manufacturer’s instructions. Cells grown in six-well plates were homogenated in buffer containing 200 µL ordinary RIPA lysis (Solarbio, Beijing, China), 0.2 µL PMSF (Solarbio, Beijing, China), and 0.2 µL protease inhibitor cocktail (Boster Biological Technology, Wuhan, China). Start the assay by mixing 20 µL of lysate with 10 µL of 5 mM Gly-Pro-pNA and then read the plate record data immediately at 2 min intervals for a total of 60 min. DPP-4 activity was calculated according to the released pNA by measuring the absorbance at 405 nM with a standard curve of p-nitroanalide. The enzyme activity was defined as the amount of enzyme catalyzing the formation of p-nitroanalide/s.
6
Oxidative Stress Markers in Stomach Tissue
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One gram of stomach tissue was rinsed for two minutes in an ice-cold solution of phosphate buffered saline (PBS) (pH 7.4, containing 0.16 mg/mL heparin). The tissue was homogenized in 10 mL of ice-cold 50 mM potassium phosphate buffer (pH 7.5, containing 1% protease inhibitor cocktail, Boster Biological Technology, Pleasanton, CA, USA, catalogue number: AR1182). The homogenate was centrifuged for 15 min at 4000 rpm at 4 °C. The total protein contents of the supernatant were measured spectrophotometrically using the Bradford method [15 (link)]. The level of lipid peroxidation products represented by malondialdehyde (MDA) in the tissue supernatant, as a marker of oxidative stress, was measured according to the colorimetric method of Tappel and Zalkin [16 (link)] and expressed as µmol/mg protein. The antioxidant markers were evaluated through the measurement of the reduced glutathione (GSH) level according to the method described by Ellman [17 ] and expressed as µmol/mg protein; and the glutathione peroxidase (GPx) activity was measured as described in the method of Paglia and Valentine [18 (link)] and expressed as U/mg protein.
7
Western Blot Analysis of BMP Signaling
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Cells were collected and lysed using a RIPA buffer (Boster Biological Technology) containing protease inhibitor cocktail and PMSF (Boster Biological Technology). After centrifugation (12,500 rpm/15 min), separate proteins were collected from cellular debris. Bicinchoninic acid method was used to determine the protein concentration. Proteins (30 μg) were separated under 90 V by voltage via the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride membrane (Millipore). After blocking the membranes with TBST containing 5% skim milk for 2 h at room temperature, the membranes were incubated with primary anti‐CHRDL2, anti‐BMP‐9, BMP‐7 (Cat No: ab269586; Abcam), BMP‐4 (Cat No: ab124715; Abcam), BMP‐2 (Cat No: ab276041; Abcam), ALK1 (Cat No: 14745‐1‐AP; Proteintech), anti‐PI3K (Cat No: 60225‐1‐Ig; Proteintech), anti‐p‐PI3K (Cat No: 17366; Cell Signaling Technology), anti‐AKT (Cat No: 10176‐2‐AP; Proteintech), anti‐p‐AKT (Cat No: 66444‐1‐Ig; Proteintech), anti‐MMP‐9 (Cat No: 10375‐2‐AP; Proteintech) antibodies (all 1:1000 dilution) overnight at 4°C. After washing with TBST three times, the membranes were incubated with the secondary antibody (dilution 1:3000) for 2 h at room temperature. Finally, the signals were detected by the Photoshop Image Analysis software CS3 (Adobe Systems).
8
Western Blot Protein Expression Analysis
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Cells were extracted using a protein lysis buffer supplemented with protease inhibitor cocktail (Boster, China). Protein concentration was determined using a BCA kit (Boster, China). Then, samples were loaded at 20–30 μg/lane and separated on a 10% precast SDS–PAGE gel, followed by transfer onto PVDF membrane (Millipore, USA). After blocking with 5% skimmed milk powder (Sigma–Aldrich, USA) for 3 h, the membrane was incubated overnight at 4 °C with the appropriate primary antibody: against PPARγ (A0270, Abclonal, China), β-actin (AC038), FABP4 (A0232), CEBP-α (A0904), MYOZ2 (A6468), or AHCY (10,757, Proteintech, China). Then, IRDye® 800CW rabbit secondary antibodies (926-32,211, LI-COR, USA) were used to detect the primary antibodies, and the membrane was imaged using a far-infrared light scanning system (LI-COR, USA).
9
Protein Extraction and Immunoblotting Protocol
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For obtaining proteins, tissues and cells were exposed to RIPA lysis buffer (Beyotime, Shanghai, China) and a protease inhibitor cocktail (Boster, Wuhan, China). Immunoblotting analysis was performed according to standard using primary antibodies containing ESR1 (A12976; Abclonal, Wuhan, China) and β-actin (AC026; Abclonal). Samples of each protein were separated by 8–12% SDS denatured polyacrylamide gel and then transferred onto a nitrocellulose (NC) membrane. The membranes were blocked for 1 h by 5% skim milk and incubated with antibody at 4 ℃ overnight. Next, the membranes were incubated with goat anti-rabbit IgG (H+L)-HRP (Sungene, Tianjin, China) as the secondary antibody (dilution, 1:3,000–5,000). The Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific, USA) was used for visualizing protein bands. ImageJ was used for analysis.
10
Western Blot Analysis of Liver Proteins
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The liver tissue was crushed and cleaved in RIPA lysis buffer containing a Protease Inhibitor Cocktail (Boster, Wuhan, China) and PhosSTOP (Roche, USA). The protein samples were separated by 12% or 15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was then incubated with specific antibodies for 10 h and with secondary antibodies (Table S2 ) at room temperature for 1 h before imaging with the ChemiDoc XRS + system (Bio-Rad Laboratories, USA). The signal intensity was quantified using Image J software.
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