Western blot was performed as previously described [70 (link)]. Cells were lysed in an extraction buffer containing 0.5% NP-40, 2 mM phenylmethanesulfonylfluooride (PMSF), 140 mM NaCl, 10 mM Tris (pH 7.0), 0.2 mM leupeptin and 0.05 mM pepstatin A. Equal amounts of protein samples were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Pall Corporation, Washington, NY, USA). After 1 h blocking with 5% non-fat milk-containing TBST buffer, proteins were recognized by incubation for 2 h with specific primary antibodies, followed by incubation with secondary antibodies conjugated with horse radish peroxidase for another 1 h. An enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA) was used to detect immunoreactivity as per the manufacturer’s instructions. Quantitative data were obtained using a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP, Upland, CA, USA).
Biospectrum ac system
Manufactured by Analytik Jena
Sourced in United States
The Biospectrum AC System is a versatile laboratory equipment designed for a range of spectroscopic applications. It features a high-performance detector and a comprehensive software interface to provide reliable and accurate measurements.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence detection kit, by Merck Group (2 mentions)
Nitrocellulose membrane, by Pall Corporation (2 mentions)
Nc membrane, by Pall Corporation (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti occludin, by Santa Cruz Biotechnology (2 mentions)
Anti zo 1, by Santa Cruz Biotechnology (2 mentions)
Ab109125, by Abcam (1 mentions)
Ab108327, by Abcam (1 mentions)
Ab133528, by Abcam (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
Phosphor creb, by Cell Signaling Technology (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Total creb, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Complete mini tablet, by Roche (1 mentions)
10 protocols using biospectrum ac system
1
Quantitative Western Blot Analysis
2
Protein Expression Detection by Western Blot
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The total protein of the cell lysates was extracted after treatment with different conditions. A detailed description of the preparation is presented in our previous report [29 (link)]. The primary antibodies used to detect specific proteins were β-actin (A544, Sigma), LC3 (PM036, Medical and Biological Laboratories, Nagoya, Japan), TIMP1 (ab109125, Abcam, Cambridge, UK), Rab37 (LTK BioLaboratories, Taiwan), ATG5 (ab108327, Abcam), and ATG7 (ab133528, Abcam). Samples were incubated overnight at 4 °C. The membranes were incubated with the secondary anti-rabbit (Amersham Pharmacia, Piscataway, NJ, USA) or anti-mouse (Chemicon, Temecula, CA, USA) antibody at room temperature for 1 h. Finally, the membrane was rinsed with enhanced chemiluminescence (ECL) (WBKLS0500; Millipore) and exposed using the BioSpectrum AC system (101-206-009; UVP, Upland, CA, USA).
3
Protein Immunoblotting Using SDS-PAGE
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Cells were harvested in lysis buffer (10 mmol/L Tris [pH 7.0], 140 mmol/L NaCl, 0.5% NP‐40, 0.2 mmol/L leupeptin, 0.05 mmol/L pepstatin A and 2 mmol/L PMSF). Equal amounts of protein samples were subjected to SDS‐PAGE and transferred onto a nitrocellulose membrane (Pall Corporation). After blocking in a 5% non‐fat milk‐containing blocking buffer for 1 hour, proteins were recognized using specific primary antibodies followed by horseradish peroxidase‐conjugated secondary antibodies. The enhanced chemiluminescence was employed to detect immunoreactivity according to manufacturer's instructions. A computing densitometer with a scientific imaging system (Biospectrum AC System, UVP) was used to obtain the quantitative data.
4
Western Blot Protein Detection Protocol
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Cells were harvested in a lysis buffer containing 10 mM Tris (pH 7.0), 140 mM NaCl, 0.5% NP-40, 0.05 mM pepstatin A, 0.2 mM leupeptin and 2 mM PMSF. Equal amounts of protein samples were subjected to SDS-PAGE and transferred onto an NC membrane (Pall Corporation, Washington, NY, U.S.A.). After blocking in a 5% non-fat milk-containing blocking buffer for 1 h, proteins were recognized using specific primary antibodies for 2 h, followed by horseradish peroxidase-conjugated secondary antibodies for another 1 h. To detect immunoreactivity, the enhanced chemiluminescence detection kit (Millipore, Billerica, MA, U.S.A.) was employed as per the manufacturer’s instructions. Quantitative data was obtained using a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP).
5
Protein Extraction and Western Blot Analysis
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After treatment, cells were harvested in lysis buffer [0.5% NP-40, 140 mM NaCl, 10 mM Tris (pH 7.0), 0.05 mM pepstatin A, 2 mM PMSF and 0.2 mM leupeptin]. Cell lysate with equal amounts of protein were subjected to SDS-PAGE and transferred onto a NC membrane (Pall Corporation, Washington, NY, U.S.A.). After transfer, membrane was incubated with 5% non-fat milk-containing blocking buffer for 1 h. Specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies were used to recognize target proteins. Enhanced chemiluminescence was used to detect immunoreactivity according to manufacturer's instructions. To obtain the quantitative data, a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP) was used.
6
Protein Expression Analysis of Keap1-Nrf2-AKT-CREB Pathway
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Tissue samples were homogenized with ice-cold lysis buffer (21 g urea, 7.6 g thiourea, 2 g CHAPS, 0.002% bromophenol blue, 150 mg DTT, and a protease and phosphatase inhibitor cocktail) using a ultrasonic machine for 10 min and centrifuged at 17,500 rpm for 1 h at 4°C) to collect the protein concentration of the supernatant. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. After the protein transfer membranes were blocked with 4% bovine serum albumin for 30 min at room temperature and the membranes were individually incubated overnight with the following primary antibodies: Keap1 (1:500, CST, Danvers, MA, USA), total-Nrf2 (1:1000, Abcam, Cambridge, England), phospho-Nrf2 (1: 500, Singalway Antibody, Baltimore, USA), BDNF (1:1000, Abcam, Cambridge, England) , total-AKT (1:1000, Cell Signaling Technology, Danvers, MA, U.S.A.), phosphor-AKT (1:1000, Cell Signaling Technology, Danvers, MA, U.S.A.), total-CREB (1:1000, Cell Signaling Technology, Danvers, MA, U.S.A.), and phosphor-CREB ((1:1000, Cell Signaling Technology, Danvers, MA, U.S.A.) at 4°C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (1:3000) for 1 h at room temperature. After washing, the protein signals were analyzed using the UVP Biospectrum AC system (UVP, USA).
7
Immunoblot Analysis for Protein Detection
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Immunoblot analyses were performed as described previously [61 (link)]. Briefly, cells were lysed in an extraction buffer containing 10 mM Tris (pH 7.0), 140 mM NaCl, 2 mM PMSF, 5 mM DTT, 0.5% NP-40, 0.05 mM pepstatin A, and 0.2 mM leupeptin. Samples of equal amounts of protein were subjected to SDS-PAGE and transferred onto a NC membrane which was then incubated in a TBST buffer containing 5% non-fat milk. Proteins were visualized by incubating with specific primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was detected based on enhanced chemiluminescence per the instructions of the manufacturer. Quantitative data were obtained using a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP).
8
Intestinal Tight Junction Protein Analysis
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Intestine tissues were homogenized and the membranes were incubated with anti-occludin (Santa Cruz Biotechnology, Inc., CA), anti-ZO-1 (750; Santa Cruz Biotechnologies, Inc.), or anti-β-actin (1:1000; Santa Cruz Biotechnologies, Inc.) Protein bands were detected using BioSpectrum AC System (UVP, Upland, CA).
9
Western Blot Analysis of Ob Protein in Frozen Hippocampus
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Frozen brain hippocampal samples were lysed with an RIPA buffer and centrifuged at 1500 rpm for 10 min. Protein concentrations were measured using a BCA protein assay kit (Sigma, St. Louis, MO, USA), and protein samples (25 μg) were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinyl difluoride (PVDF) membranes, and blocked with 5% non-fat milk in Tris-buffered saline, Tween-20 (TBST) buffer. Membranes were incubated for 8 h at 4 °C with primary antibodies: rabbit polyclonal anti-Ob (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse monoclonal anti-β actin (1:1000; Santa Cruz Biotechnology). Membranes were subsequently incubated with anti-rabbit or anti-mouse antibodies for 1 h at room temperature and then reacted with enhanced chemiluminescence reagents. Signals were detected by the UVP Biospectrum AC System (UVP, Upland, CA, USA) and analyzed using the Image-pro Plus software.
10
Intestinal Tight Junction Protein Analysis
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Intestinal tissues were homogenized in ice-cold buffer containing 50 mmol/L Tris•HCl (pH 7.5), 1 mmol/L ethylene glycol tetraacetic acid, 1 mmol/L ethylenediaminetetraacetic acid, and a protease inhibitor cocktail (cOmplete Mini tablets; Roche, Mannheim, Germany). Proteins (30 µg) were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis subject to reducing conditions and electroblotted to a polyvinylidene di uoride membrane (Immobilon-P, Millipore, Millipore Merck Corporation, MA, USA). After the membranes were blocked with 5% nonfat dry milk, they were incubated with antioccludin (Santa Cruz Biotechnology, Inc., CA, USA), anti-ZO-1 (750; Santa Cruz Biotechnologies, Inc.), or anti-β-actin (1:1000; Santa Cruz Biotechnologies, Inc.). Protein bands were detected using the BioSpectrum AC System (UVP, Upland, CA, USA).
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