For extraction of the total protein, left ventricular heart tissues from rats were homogenized in commercial RIPA buffer (Boster, Wuhan, China) added with protease inhibitor and phosphatase inhibitor (MedChemExpress, America). Then, the supernatants were obtained after centrifugation at 12000×g at 4°C for 20 min. The protein concentration was detected using a bicinchoninic acid assay (Boster, Wuhan, China). Denatured proteins (40 μg/lane) were loaded on and separated by 8-10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred onto polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% BSA at room temperature for 1 h, which subsequently were incubated with primary antibodies overnight at 4°C. After washing with TBS-T, the membranes were probed with peroxidase-conjugated secondary antibodies (Boster, Wuhan, China) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit following the manufacturer's instructions (New Cell & Molecular Biotech, Suzhou, China). Protein expression contents were analyzed by ImageJ software, and the level of β-actin or GAPDH was used as an internal control. The primary and secondary antibodies used in this study were listed in Supplementary Table 1 .
Peroxidase conjugated secondary antibody
Manufactured by Boster Bio
Sourced in China
Peroxidase-conjugated secondary antibodies are laboratory reagents used in various immunoassay techniques. They function as detection tools, binding to primary antibodies and catalyzing a colorimetric reaction to visualize target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Protease inhibitor and phosphatase inhibitor, by MedChemExpress (1 mentions)
Bicinchoninic acid assay, by Boster Bio (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
β actin, by Boster Bio (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Anti β actin, by Boster Bio (1 mentions)
Anti robo1, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
0.22 μm polyvinylidene fluoride, by Merck Group (1 mentions)
Mouse β actin, by Cell Signaling Technology (1 mentions)
Mouse active β catenin, by Merck Group (1 mentions)
Ecl kit, by Cytiva (1 mentions)
12 protocols using peroxidase conjugated secondary antibody
1
Protein Extraction and Western Blot Analysis
2
Quantitative Western Blot Analysis
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HK-2 cells and kidney tissue were lysed, and total proteins were extracted with Cell Lysis Buffer for Western and IP (Beyotime, China). The protein concentration was then adjusted to 35 µg/µl with an Enhanced BCA Protein Assay Kit (Beyotime, China). The protein samples were subjected to 10% SDS-PAGE separation and then transferred to PVDF membranes. The PVDF membranes were incubated with antibodies against PDPK1 (1 : 1000, Absin, 0006890201), AKT (1 : 1000, CST, 4691T), p-AKT (1 : 1000, CST, 4060), and β-actin (1 : 1000, Boster). Then, the membranes were incubated with peroxidase-conjugated secondary antibodies (1 : 10000, Boster). The protein bands were visualized by enhanced chemiluminescence, and the protein expression was quantitatively analysed by ImageJ.
3
Immunoblotting of Cellular Proteins
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Total cellular lysates were obtained by lysing cells in radioimmunoprecipitation assay buffer (RIPA buffer) containing protease and phosphatase inhibitors (Roche, Mannheim, Germany). The concentration of proteins was determined using BCA Protein Assay Kit (Thermo Scientific). SDS-PAGE was performed, and then proteins were blotted on nitrocellulose membrane. The membrane was blocked with a blocking solution (TBST+5%BSA) for 1 h and incubated with the primary antibody for 2 h. After washing for three times, the membrane was immersed with appropriate peroxidase-conjugated secondary antibodies (Boster). Detection of proteins was conducted by using ECL reagent (Thermo Scientific).
4
Retinal Protein Extraction and Immunoblotting
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Protein extracts from retinal tissues were prepared at different time points on ice using a protein extraction kit and protease inhibitor kit (Pierce, Rockford,IL). Protein extracts were sonicated in 500ul lysis buffer. The homogenates were centrifuged at 12,000 g for 20 min at 4°C. The supernatants were collected, transferred to a fresh tube and stored at -80°C in preparation for SDS-PAGE(sodium dodecyl sulfate-Polyacrylamide gel electrophoresis). The protein content of each lysate was measured using a BCA Protein Assay Kit (Tianlaishengwu, tianlai, China) according to the manufacturer’s directions with BSA as the standard (Bio-Rad Laboratories, Hercules, CA). The samples were then boiled for 5 min in sample buffer. Equal amounts of protein were loaded and analyzed by immunoblotting. The primary antibodies anit-Slit2 (1:1000; Millipore, Temecula, CA), anti-Robo1 (1:500; Abcam, Cambridge, UK) and anti-β-actin (1:1000; Boster, China) were used. Membranes were washed and incubated with peroxidase-conjugated secondary antibodies (1:6000; Boster, China), Western blot image was analyzed by a tool called “Image J”. The band densities of the Slit2 and Robo1 proteins were normalized to each β-actin internal control. Western blots were repeated three times, and qualitatively similar results were obtained.
5
Protein Expression Profiling of hRPR Cells
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The hRPR cells were washed three times with ice-cold PBS (4°C, pH 7.4) for 5 min at room temperature and prepared using a protein extraction kit and a protease inhibitor kit (Pierce, Rockford, IL). After centrifugation, the supernatant was collected, and the protein content of each lysate was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce) according to the manufacturer’s instructions. Equal amounts of protein (40 μg) were separated by a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a 0.22-μm polyvinylidene fluoride (PVDF) membrane (Millipore). The primary antibodies used to probe the membranes included anti-UBE3D (1:500, PAB21883, Abnova, USA), anti-CyclinB1 (1:1,000,# 4138, CST, USA), anti-caspase-3 (1:500, ab13847, Abcam, Cambridge, MA, USA), anti-p38 mitogen-activated protein kinases (p38MARK) (1:1,000, #8690, CST, USA), anti-phosphorylated p38 MAPK (p-p38MARK) (1:1,000, #4511, CST, USA), anti-P62 (1:2,000, PM045, MBL, Japan), anti-light chain3 (LC3) (1:1,000, #2775, CST, USA), anti-Beclin1 (1:2,000, ab207612, Abcam, Cambridge, MA, USA) and anti-β-actin (1:2,000, #4970 CST, USA). The membranes were washed and incubated with peroxidase-conjugated secondary antibodies (1:5,000, Boster, China). The proteins were visualized with enhanced chemiluminescence western blot detection reagents (Millipore).
6
Western Blot Analysis of Signaling Proteins
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Cells were harvested in RIPA lysis buffer (Beyotime Co.). Whole-cell protein extracts were quantified by the BCA assay, separated on 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk powder in PBST for 3 h. The membranes were probed overnight with the primary antibodies and then 4 h with peroxidase-conjugated secondary antibodies (Boster, Wuhan, China). Antibodies used in this study included the mouse β-Actin, rabbit Smad1, rabbit phospho-Smad1, mouse GSK3β, rabbit phospho-GSK3β (Cell Signaling, Beverly, MA, USA), rabbit β-catenin (Abcam, Cambridge, MA, USA) and mouse active β-catenin (Millipore). The blots were visualized using an ECL Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's recommended instructions. To measure the protein abundance, gray value of the blots in scanned images was measured with ImageJ Plus software (National Institutes of Health, Bethesda, MD, USA). Gray value of each target protein was normalized to that of β-Actin before comparison.
7
Protein Expression Analysis by Western Blot
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Total proteins were extracted and solubilized in a RIPA buffer containing proteinase inhibitors (1% cocktail and 1 mmol/L phenylmethylsulfonyl fluoride, Sigma-Aldrich, USA). After the determination of protein concentration with a BSA assay kit (Pierce, Rockford IL, USA), 40 μg of total protein from each sample was separated by 10% SDS-PAGE at 70 V for 30 min, followed by 110 V until the end and transferred to PVDF membranes (Millpore, USA). The membranes were incubated with the primary antibodies overnight at 4°C (Abcam Technology, UK: HIF-1α, 1:2,000; claudin-1, 1:4,000; occludin, 1:1,500; GAPDH, 1:2,000; Thermo Fisher Scientific, USA: ZO-1, 1:1,000), followed by incubation with the peroxidase-conjugated secondary antibodies (Boster Biological Technology, Wuhan, China, 1:5,000) at room temperature for 1 h. The protein bands were detected with the enhanced chemiluminescence (ECL) detection system (Pierce, Rockford IL, USA) and quantitated by densitometry using Image J Software.
8
Western Blot Analysis of Protein Expression
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The western blot analyses were performed as previously described (Liao et al., 2013 (link)). The proteins were loaded on 10% sodium dodecyl sulphate polyacrylamide gels, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk powder in PBST (containing 0.1% Tween). The membranes were probed overnight with the following primary antibodies: β-actin (Cell Signalling, Beverly, MA, USA), homeodomain-containing factor A10 (HOXA10) (Santa Cruz, Dallas, TX, USA) and Forkhead box O1 (FoxO1) (Cell Signalling). The membranes were incubated with peroxidase-conjugated secondary antibody (Boster, Wuhan, China). The blots were visualized through an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA) according to the recommended instructions.
9
Western Blot Analysis of PBMC Proteins
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PBMC proteins were extracted using RIPA lysis buffer with a proteinase inhibitor. The protein concentration in the lysates was measured by the BCA protein assay kit (#23227, Pierce, ThermoFisher), and 50 μg of the total protein mixed with 4x SDS loading buffer was loaded per lane. The proteins in the lysates were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). In order to block nonspecific binding, the PVDF membranes were incubated with 5% skim milk powder at room temperature for 1 h. The PVDF membranes were then incubated for 12 h at 4°C with an antiserum containing antibodies against IL-6 (#12912, Cell Signaling Technology), Stat3 (#9139, Cell Signaling Technology), Phospho-Stat3 (Tyr705) (#9145, Cell Signaling Technology), and GAPDH (MA5-15738, Invitrogen). Primary was diluted at 1 : 1,000. A peroxidase-conjugated secondary antibody (1 : 5,000 dilution, BOSTER Biological Technology Co. Ltd, Wuhan, China) and enhanced chemiluminescence western blot detection reagents (NCI4106, Pierce, ThermoFisher) were used to visualize the target proteins, which were quantified with ChemiDoc XRS+ (Bio-Rad Laboratories, Inc.).
10
Western Blot Analysis of Signaling Proteins
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Cells were washed twice with PBS and harvested in RIPA buffer. The nuclear proteins were extracted using NE-PER(R) Nuclear and Cytoplasmic Extraction kit (Thermo). Total cellular or nuclear extracts were separated with SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat milk in Tris-buffered saline for 1 h at room temperature, the membrane was incubated with primary antibodies against AP-1 (Santa Cruz Biotechnology), RIG-I, TLR3, NF-κB, ERK, phospho-ERK, p38MAPK, and phospho-38MAPK (Cell Signaling Technology). After washing with Tris-buffered saline containing 0.5% (w/v) Tween [18 (link)], the membrane was incubated with an appropriate peroxidase-conjugated secondary antibody (Boster). Each blot was developed using SuperSignal West Pico and SuperSignal West Femto (Thermo). The images were captured using the MF-Chemi Bio Imaging System (DNR). To correct the results for potential variations in sample load, each blot was stripped and reprobed with anti-β-tubulin or anti-GAPDH (Santa Cruz Biotechnology). The fold changes of protein bands were analyzed with Image J software.
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