Total protein was extracted from right lung tissue from rats and PASMC cultures treated or transfected as described above, and resulting protein concentrations were assayed using the BCA method. Equal amounts of protein (20 μg) were electrophoresed on an SDS/polyacrylamide gel (12% gel), and then electroblotted on to PVDF membrane. Membranes were blotted with primary antibodies (CST, U.S.A.) against t-p38 MAPK (1:2000), p-p38 MAPK (1:1000), t-ERK MAPK (1:2000), or p-ERK MAPK (1:1000). As a loading control, membranes were blotted with antibody against tubulin (1:2000; Epitomics, U.S.A.). Then membranes were blotted with a horseradish peroxidase–conjugated secondary antibody (1:4000; Epitomics, U.S.A.) and stained using the Clarity ECL Western Blot Substrate (Bio–Rad, U.S.A.).
Horseradish peroxidase conjugated secondary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Horseradish peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody that is chemically conjugated to the enzyme horseradish peroxidase. This conjugate can be used to detect and amplify the signal of a primary antibody that has bound to a target antigen in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (33 mentions)
β actin, by Abcam (30 mentions)
Gapdh, by Abcam (29 mentions)
Ripa lysis buffer, by Beyotime (23 mentions)
Polyvinylidene difluoride membrane, by Merck Group (19 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Anti gapdh, by Abcam (16 mentions)
Bca protein assay kit, by Beyotime (15 mentions)
Polyvinylidene fluoride membrane, by Merck Group (15 mentions)
Anti β actin, by Abcam (14 mentions)
Protease inhibitor cocktail, by Merck Group (12 mentions)
Pvdf membrane, by Thermo Fisher Scientific (11 mentions)
Ripa buffer, by Beyotime (10 mentions)
Ripa buffer, by Merck Group (9 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (9 mentions)
E cadherin, by Abcam (9 mentions)
Nitrocellulose membrane, by Merck Group (9 mentions)
Anti e cadherin, by Abcam (9 mentions)
Enhanced chemiluminescence kit, by Cytiva (9 mentions)
Anti n cadherin, by Abcam (9 mentions)
374 protocols using horseradish peroxidase conjugated secondary antibody
1
Western Blot Analysis of MAPK Signaling
2
Nampt Protein Expression Analysis in PC12 Cells
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After treatment, PC12 cells were washed twice with PBS. The cells were lysed in RIPA buffer produced by Millipore (Temecula, CA, USA) which contained 1 mM phenylmethanesulfonyl fluoride and 1% protease inhibitor cocktail produced by CWBio (Beijing, China). After centrifugation at 12,000 rpm for 10 min at 4 °C, the protein concentrations of the samples were determined by BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Thirty μg of the sample was electrophoresed through a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.45-μm nitrocellulose membranes. The blots were incubated with a polyclonal Nampt antibody from Abcam (Cambridge, UK), which was diluted in TBST containing 1% bovine serum albumin at 1:2,000 ratio, at 4 °C overnight. Subsequently, a horse radish peroxidase-conjugated secondary antibody produced by Epitomics (Zhejiang Province, China) diluted by TBST containing 1% bovine serum albumin at 1:3,000 ratio was used for incubation of the blots for 1 h at room temperature. The normalization of the sample was conducted by using a Tubulin antibody produced by proteintech (Wuhan, China). Quantifications of the bands were performed by Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD, USA).
3
Quantification of Liver Protein Extraction
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Protein of liver samples was extracted and quantified by using a Bradford protein assay kit (Bio-Rad). Briefly, liver samples were homogenised at 48C (1:10, w/v) in 50mM-Tris-HCl buffer (pH ¼ 8•0), containing 1 mmol/l phenylmethylsulfonyl fluoride, 1 mg/ml protease inhibitor cocktail, 0•1 % b-mercaptoethanol, and centrifuged at 12 000 g at 48C for 5 min. After being heated to 958C for 5 min, proteins were separated on SDS-PAGE gels, and then transferred onto immobilon-P polyvinylidene difluoride membranes (Millipore). After blocking with 5 % non-fat milk/Tween overnight, the membranes were immunoblotted with the antibodies specifiedanti-FGF-21 antibody (Epitomics), and a horseradish peroxidase-conjugated secondary antibody (KPL) was used in the detection of specific proteins. For examining the equal loading, b-actin antibody (Santa Cruz Biotechnology) was used as control. Finally, enhanced chemiluminescence reagent (Amersham) was used to visualise the protein bands. Band intensities were determined by using AlphaEase FC analysis software (version 4; Alpha Innotech).
4
Western Blot Analysis of Protein Signaling
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Total protein extracts were resolved on a 10% SDS-PAGE and transferred onto a Hybond PVDF membrane (GE Healthcare, Little Chalfont, UK). Then, the membranes were probed with primary antibodies, followed by the incubation with horseradish peroxidase-conjugated secondary antibody (Abcam; dilution 1 : 10 000). The protein bands were quantified with the enhanced chemiluminescence solution (ECL, GE Healthcare) with an ImageJ software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies were used: anti-MMP-9 (Abcam; dilution 1 : 1000), anti-MMP-2 (Abcam; dilution 1 : 2000), anti-Bcl-2 (Cell Signaling Technology, Danvers, MA, USA; dilution 1 : 1000), anti-cleaved caspase-3 (Cell Signaling Technology; dilution 1 : 1000), anti-Bax (Abcam; dilution 1 : 5000), anti-PI3K (Abcam; dilution 1 : 1000), anti-mTOR (Cell Signaling Technology; dilution 1 : 1000), anti-AKT (Abcam; dilution 1 : 10 000), anti-p-PI3K (Abcam; dilution 1 : 1000), anti-p-mTOR (Cell Signaling Technology; dilution 1 : 1000) and anti-p-AKT (Abcam; dilution 1 : 500). β-Actin was used as a protein control.
5
Protein Expression Analysis in HCC Cells
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Total protein was extracted from HCC cells by using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein content was measured with a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were electrophoresed on 10% SDS-PAGE gels and transferred onto PVDF membranes. Then, the membranes were incubated with the primary antibodies against E-cadherin (Cat#14,472; 1:1,000 dilution), Vimentin (Cat#12,826; 1:1,000 dilution), KPNA2 (Cat#14,372; 1:1,000 dilution), p53 (Cat#48,818; 1:1,000 dilution), p21 (Cat#2947; 1:1,000 dilution), Bax (Cat#5023; 1:1,000 dilution), Cyclin D1 (Cat#2922; 1:1,000 dilution) and GAPDH (Cat#5174; 1:1000 dilution) overnight at 4°C, followed by probed with horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, MA, USA; 1:2000; Cat#ab6721) for 2 h at room temperature. The immunoreactive bands were detected by ECLWestern Blotting Detection System (GE Healthcare, Chicago, IL, USA). All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Quantification of COX2 Protein Expression
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The experiments were performed as previously described (Wang et al., 2011 (link); Ding et al., 2017 (link)). The protein content was extracted from tissue and cell samples by using a RIPA lysis buffer (Invitrogen, Waltham, MA, United States) based on the protocol provided by the assay kit manufacturer. In the next step, the concentration of protein lysate was quantitatively evaluated by using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States) based on the protocol provided by the assay kit manufacturer, and an equal amount of the protein lysate from each sample was separated on a 10% sodium dodecyl sulfate–polyacrylamide gel. Then, the separated proteins were blotted to a polyvinylidene fluoride membrane, which was then blocked with 5% serum and incubated in sequence with primary anti-COX2 antibody, as well as horseradish peroxidase–conjugated secondary antibody (Abcam, Cambridge, CA, United States) based on the protocol provided by the antibody manufacturer. The signal of protein band was detected by utilizing an ECL chemofluorescence reagent (Pierce, Rockford, IL, United States) based on the protocol provided by the reagent manufacturer to calculate the relative expression of COX2 protein in each sample using the expression of β-actin protein as the control.
7
Apoptosis Protein Expression Analysis
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The proteins were extracted from the cells using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and their concentration levels were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The proteins (40 µg per lane) were separated by SDS-PAGE on 10% gels, and were transferred onto 0.45 µm PVDF membranes (Thermo Fisher Scientific, Inc.). Following blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Anti-Bax (Abcam; cat. no. ab32503; 1:1,000), anti-Bcl-2 (Abcam; cat. no. ab32124; 1:1,000), anti-cleaved caspase 3 (Abcam; cat. no. ab2302; 1:1,000), anti-cytochrome c (Cyto C; Abcam; cat. no. ab13575; 1:1,000), anti-apoptotic protease activating factor-1 (Apaf 1; Abcam; cat. no. ab2001; 1:1,000), anti-cleaved caspase 9 (Abcam; cat. no. ab2324; 1:1,000) and anti-β-actin (Abcam; cat. no. ab8227; 1:1,000). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Abcam; cat. no. ab150077; 1:200) for 1 h at room temperature. Finally, an image of the protein band was detected using ECL reagent (Santa Cruz Biotechnology, Inc.). Image-Pro Plus software (version 7, Media Cybernetics, Inc.) was used for the targets that were normalized to β-actin.
8
Western Blot Analysis of Key Cellular Proteins
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Kidney or spleen tissues and CD4+T cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific, USA). Protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE), then transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen, USA). Blots were incubated with primary antibodies against ROCK2 (1:500; Abcam, USA), p-STAT3 (1:2000; Abcam, USA), p-STAT5 (1:500; Abcam, USA), Calnexin (1:2000; Abcam, USA), CD63 (1:500; Abcam, USA), Alix (1:1000; Abcam, USA), TSG101 (1:2000; Abcam, USA), β-actin (Sigma, USA) and horseradish peroxidase-conjugated secondary antibody (Abcam, USA). β-actin was used as an internal control. Blots were visualized by Molecular Imager® Gel Doc™ XR System (Bio-Rad, USA).
9
Western Blot Analysis of IER3 Protein
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PFMCs and CD4+ T cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, China). Protein samples (50 ng) was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen, USA). The membrane was incubated with primary antibodies against IER3 (Invitrogen, USA), β-actin (Abcam, USA) and horseradish peroxidase-conjugated secondary antibody (Abcam, USA). Blots were detected by enhanced chemiluminescence, and band intensities were quantified using image software Image Lab (Bio-Rad, USA). β-actin was used as an internal control.
10
Western Blot Analysis of Sirt4 Protein
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Cardiomyocytes were collected and lysed after which equivalent quantity of protein was loaded and separated by 10% SDS-PAGE. Then all the separated protein was electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membrane was blocked by 5% skim milk for 1h, incubated with primary antibodies (Sirt4, 1:1000, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA; GAPDH, 1:2000, Cell Signaling Technology, Boston, USA) at 4°C overnight. Afterwards horseradish peroxidase-conjugated secondary antibody (1:10,000, Abcam, LA, USA) was also incubated on the membrane at 37°C for 1 h. Proteins were scanned and detected by enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) using a ChemiDoc MP system (Bio-Rad Laboratories). Image J software was used to analyze densitometric results of band.
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