Mouse anti-MMP2, mouse anti-PP2Ac, and mouse anti-B2M antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Total protein was extracted using a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and supplemented with protease inhibitor cocktail kit (Roche). The protein extract was loaded onto an SDS-polyacrylamide gel, size-fractionated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, CA, USA). After blocking in 5% non-fat milk for 1 hour, the membranes were incubated overnight with primary antibodies at 4 °C. The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence (ECL, Millipore, St Charles, MO, USA) detection. The intensity of the bands was captured by JS-1035 image analysis scanning system (Peiqing Science & Technology, Shanghai, China). B2M was used as the internal control.
Protease inhibitor cocktail kit
Manufactured by Roche
Sourced in Switzerland
The Protease inhibitor cocktail kit is a laboratory product designed to inhibit the activity of proteases, which are enzymes that break down proteins. The kit contains a pre-formulated mixture of protease inhibitors that can be added to cell or tissue samples to prevent unwanted protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemi luminescence (ecl), by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Mouse anti mmp 2, by Santa Cruz Biotechnology (1 mentions)
Mouse anti pp2ac, by Santa Cruz Biotechnology (1 mentions)
Mouse anti b2m, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Rabbit anti β actin, by Proteintech (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Lmp2a, by Abcam (1 mentions)
Horseradish peroxidase hrp linked secondary antibody, by Jackson ImmunoResearch (1 mentions)
β actin, by Sino Biological (1 mentions)
Uvp biospectrum 500 imaging system, by Analytik Jena (1 mentions)
Atg12, by Cell Signaling Technology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti γ tubulin, by Merck Group (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Hif 1α, by Thermo Fisher Scientific (1 mentions)
6 protocols using protease inhibitor cocktail kit
1
Quantifying Protein Expression via Western Blot
2
Western Blot Analysis of β-Catenin
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Total protein was extracted using a lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1 mM EDTA) supplemented with a protease inhibitor cocktail kit and a phosphatase inhibitor cocktail kit (Hoffman-La Roche Ltd., Basel, Switzerland). The protein extracts were loaded, size-fractionated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). After blocking, the membranes were incubated with the primary antibodies mouse anti-β-catenin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit anti-β-actin (Proteintech Group Inc., Chicago, IL, USA) at 4°C overnight. Protein expression was determined using horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies, followed by detection using enhanced chemiluminescence (EMD Millipore). Band intensity was visualized using a JS-1035 image analysis scanning system (Shanghai Peiqing Science & Technology, Co., Ltd., Shanghai, People’s Republic of China).
3
Protein Expression Analysis in Tissue
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Total protein in tissue and cells was extracted by homogenizing sample in ice-cold radioimmunoprecipitation assay buffer with protease inhibitor cocktail kit (Roche). The lysates were centrifuged and the supernatants were collected for measurements of protein concentrations using a bicinchoninic acid assay reagent kit. Fifty microgram of total protein was loaded to run 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was transferred to nitrocellulose membranes (GE Healthcare Biosci). Then, the membranes were incubated for 30 minutes in 5% low-fat milk prepared with Tris-buffered saline containing 0.05% Tween-20 (TTBS). Subsequently, the membranes were incubated with primary antibodies as the rabbit anti-TGF-β1, anti-TRPC6, anti-nephrin, anti-desmin and anti-caspase-9 (1:200-1:500, obtained from Neuromics and Abcam Co). After being fully washed, the membrane was incubated with horseradish peroxidase-linked anti-rabbit secondary antibody (1:250) and visualized for immunoreactivity. The membrane was also processed to detect β-actin or GAPDH for equal loading. The bands recognized by the primary antibody were visualized and the optical densities of protein bands were analyzed using the ImageJ software.
4
Western Blot Analysis of Protein Expression
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CNE-2 cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and supplemented with protease inhibitor cocktail kit (Roche Biochemicals, Penzberg, Germany). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membrane was incubated with primary antibodies for LMP1- (1:2000, Abcam, Cambridge, UK), LMP2A- (1: 1000, Abcam), eIF4E-specific (1: 1000, Abcam) or β-actin (1: 3000, Sino Biological, Wayne, PA, USA). Horseradish peroxidase (HRP)-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and enhanced chemiluminescence (Thermo Fisher Scientific) were utilized to visualize the specific binding of the HRP-linked second antibody to first antibody-protein complexe, under the UVP BioSpectrum 500 imaging system (UVP, Upland, CA, USA).
5
Western Blot Protein Analysis Protocol
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Total protein was extracted using a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and supplemented with protease inhibitor cocktail kit (Roche). The protein extract was loaded onto an SDS-polyacrylamide gel, size-fractionated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, CA, USA). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight with primary antibodies at 4 °C. The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence (ECL, Millipore, St Charles, MO, USA) detection. The intensity of the bands was captured by JS-1035 image analysis scanning system (Peiqing Science & Technology, Shanghai, China). β-actin was used as the internal control.
6
Western Blot Analysis of Cellular Signaling Proteins
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Cells were lysed in RIPA buffer containing Protease Inhibitor Cocktail kit (Roche, Basel, Switzerland). Then, 20 μg of total protein was separated by SDS-PAGE and then transferred to a PVDF membrane. After blocking in 10% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C or 2 h at room temperature. The membranes were then incubated with horseradish peroxidase labeled secondary antibodies and visualized with ECL. The antibodies used and their sources are as follows: HIF-1α (1:500, MA1-516, Thermo Fisher, Waltham, MA, USA), p53Ab1 (1:1000, 9282, Cell Signaling, Beverly, MA, USA), phospho-p53 (Ser15) (1:500, 9284, Cell Signaling), LC-3 (1:5000, 2775, Cell Signaling), PARP (1:1000, 9542, Cell Signaling), caspase-3 (1:1000, 9662, Cell Signaling), GAPDH (1:1000, sc-32233, Santa Cruz, Dallas, TX, USA), Atg7 (1:1000, 8558, Cell Signaling), Atg12 (1:1000,4180, Cell Signaling), HSF1 (1:000, sc-17756, Santa Cruz), HSP70 (1:1000, Stressgen, Farmingdale, NY, USA), P-ser473 AKT (1:1000, 9271, Cell Signaling), AMPK (1:1000, 5831S, Cell Signaling), p-AMPK (1:1000, 5831, Cell Signaling), mTOR (1:1000, A2445, Cell Signaling), p-mTOR (1:1000, 2971, Cell Signaling), p62 (1:1000, 8025, Cell Signaling, Beverly, MA, USA), and anti-γ-tubulin (1:8000, t6557, Millipore, St. Louis, MO, USA).
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