Approximately 50 mg samples were minced to small pieces using surgical blades and sonicated in a protein lysis buffer containing protease inhibitors (complete, Ultra, Mini, EDTA-free, EASYpack Roche, Germany). Protein concentrations were measured by the BCA method, and samples were adjusted to the same protein concentration, aliquoted and stored at −80°C. Equal amounts of total proteins from tissues were separated on SDS-polyacrylamide gels and electro-transferred to PVDF membranes (Roche Germany). The blots were incubated with following polyclonal antibodies overnight at 4°C: anti-BMP2 (1:1000, Abcam), anti-GDNF (1:1000, Abcam), and anti-pSmad1/5/8 (1:1500, Abcam). Blots were washed and incubated with horseradish peroxidase-linked secondary antibodies (1:2000, Abcam) for 2 h at room temperature and detected using ECL reagent (Millpore, MA, United States).
Horseradish peroxidase linked secondary antibody
Manufactured by Abcam
Sourced in United States
Horseradish peroxidase-linked secondary antibodies are enzyme-conjugated antibodies used as detection reagents in various immunoassays and immunohistochemical techniques. The horseradish peroxidase enzyme catalyzes a colorimetric reaction, allowing for the visualization and quantification of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Abcam (2 mentions)
Anti bmp2, by Abcam (1 mentions)
Anti gdnf, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Chemidoc platform, by Bio-Rad (1 mentions)
Pvdf sheet, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid quantitation kit, by Beyotime (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
Sds page, by Beyotime (1 mentions)
Supersignal west femto kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
14 protocols using horseradish peroxidase linked secondary antibody
1
Protein Expression Analysis in Tissues
2
Immunofluorescence Labeling of Angiogenic Markers
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Purified rat anti-mouse CD31 monoclonal IgG2a antibody was purchased from BD Bioscience (San Diego, CA, USA). Rabbit anti-mouse vascular endothelial growth factor (VEGF) polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anticystathionine γ-lyase (CSE), beta-actin antibodies, and horseradish peroxidase-linked secondary antibodies were obtained from Abcam (San Francisco, CA, USA). Mouse VEGF ELISA kits were purchased from eBioscience (San Diego, CA, USA). Sodium sulfide standard was from Alfa Aesar (cat. number 65122, Ward Hill, MA, USA). Streptozotocin (STZ) and other chemicals frequently used in our laboratory were purchased from Sigma-Aldrich Co. (St Louis, MO, USA).
3
Protein Expression Analysis in H9C2 Cells
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Protein samples from H9C2 cells were extracted with RIPA buffer (Thermo Fisher Scientific, USA) supplemented with protease and phosphatase inhibitors. The BCA kit (Thermo Fisher Scientific, USA) measured protein concentrations. SDS‐PAGE resolved proteins, which were then blotted onto PVDF sheets (Millipore, USA). These membranes were subsequently probed using primary antibodies targeting FLRT3, SMAD4, ANP, BNP, Bcl‐2, Bax, LC3I, LC3II, SCN5A, KCNIP2, KCND2 (all 1:1000, Abcam) and β‐actin (1:5000, Cell Signalling Technology) for normalization. Following primary incubation, blots were exposed to horseradish peroxidase‐linked secondary antibodies (1:5000, Abcam). Detection was accomplished via the ECL kit (Thermo Fisher Scientific) and visualized on a ChemiDoc platform (Bio‐Rad, USA).
4
Protein Extraction and Western Blot Analysis
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Cells and ovarian tissues were lysed in radioimmunoprecipitation assay buffer with protease inhibitor cocktail and PhosSTOP (Roche, Basel, Switzerland). Protein extraction was analyzed using a bicinchoninic acid quantitation kit (Beyotime, Beijing, China). After 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane with primary antibodies, followed by horseradish peroxidase–linked secondary antibodies (Abcam, Hongkong, Chian) and enhanced chemiluminescence reagents (Millipore Corp., Billerica, MA, USA). All antibodies used are listed in Supplementary Table 2
5
Chondrocyte Protein Expression Analysis
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Rat chondrocytes were lysed in RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Beyotime). The protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Equal amounts of protein (50 µg) were separated by 10% SDS-PAGE (Beyotime) and transferred to PVDF membranes (Millipore). Following transfer, the membranes were blocked with 5% BSA in TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies (COL 2, COL 10, SOX-9, and ACAN), directly diluted at a ratio of 1:1000 in 5% BSA-TBST. The following day, after washing thrice with TBST, the membranes were incubated with horseradish peroxidase-linked secondary antibodies, directly diluted at a ratio of 1:5000 (Abcam), for 1 h at room temperature. After washing three times with TBST, the membranes were visualized using the SuperSignal West Femto Kit (Thermo Fisher). Finally, the intensity of the blots was quantified using Image Lab 3.0 software (Bio-Rad). After washing three times with TBST, the membranes were visualized using the SuperSignal West Femto Kit (Thermo Fisher). Finally, the intensity of the blots was quantified using Image Lab 3.0 software (Bio-Rad) (Huang et al., 2017 (link)).
6
Protein Expression Analysis in HDEC and CRL-2586 Cells
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HDEC and CRL-2586 EOMA cell lines were harvested and extracted using lysis
buffer, and then equal amount of cell extracts were separated on 15% sodium
dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Separated
protein bands were transferred into polyvinylidene fluoride (PVDF) membranes
from Millipore Corporation (Billerica, MA, USA), which were blocked in 5% skim
milk powder. The primary antibodies against HIF1A, PCNA, and cleaved caspase-3
were diluted according to the instructions of antibodies and incubated overnight
at 4°C. Then, horseradish peroxidase–linked secondary antibodies (Abcam,
Cambridge, MA, USA) were added at a dilution ratio of 1:1000 and incubated at
room temperature for 2 h. The membranes were washed with phosphate-buffered
saline (PBS) and the bands were visualized using ECL-PLUS/Kit (GE Healthcare,
Piscataway, NJ, USA) according to the kit’s instruction.
buffer, and then equal amount of cell extracts were separated on 15% sodium
dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Separated
protein bands were transferred into polyvinylidene fluoride (PVDF) membranes
from Millipore Corporation (Billerica, MA, USA), which were blocked in 5% skim
milk powder. The primary antibodies against HIF1A, PCNA, and cleaved caspase-3
were diluted according to the instructions of antibodies and incubated overnight
at 4°C. Then, horseradish peroxidase–linked secondary antibodies (Abcam,
Cambridge, MA, USA) were added at a dilution ratio of 1:1000 and incubated at
room temperature for 2 h. The membranes were washed with phosphate-buffered
saline (PBS) and the bands were visualized using ECL-PLUS/Kit (GE Healthcare,
Piscataway, NJ, USA) according to the kit’s instruction.
7
Western Blot Analysis of Glioma Proteins
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The proteins from glioma tissues and cells were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA), separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride membranes (PVDF, Invitrogen). After blocked with 5% skim milk powder for 2 h, the membranes were probed with primary antibodies overnight at 4 °C and maintained in horseradish peroxidase-linked secondary antibodies (1:4000, Abcam, Cambridge, MA, USA) for 1 h. Finally, the proteins were visualized by chemiluminescence and the relative protein expression was analyzed by the ImageJ software and normalized to GAPDH. The primary antibodies were myeloid cell leukemia-1 (MCL-1, 1:1000, Abcam), multidrug resistance-associated protein-1 (MRP-1, 1:500, Abcam), cyclinD1 (1:2000, Abcam), B-cell lymphoma-2-associated x (Bax, 1:2000, Abcam), caspase-3 (1:2000, Abcam), CPEB4 (1:1000, Thermo Fisher Scientific), PI3K (1:1000, Abcam), P-PI3K (1:1000, Abcam), AKT (1:1000, Abcam), P-AKT (phospho Ser473) (1:1000, Abcam), and GAPDH (1:2000, Abcam).
8
Western Blot Analysis of Protein Extracts
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Protein extracts were obtained by lysis buffer, and protein concentrations were measured using a BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, P.R. China). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked in 5% defatted milk for 1 h and then incubated with primary antibodies overnight at 4°C. Afterward, membranes were probed with horseradish peroxidase-linked secondary antibodies (1:2,000; Abcam) for 1 h. Protein bands were detected using an ECL Plus Western Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
9
Immunoblotting of Cell Signaling Proteins
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Equal total protein extracts were loaded and separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were immunoblotted with the following antibodies: anti-IRS4 (ab52622; Abcam, Cambridge, MA, USA), anti-p21 (ab109520; Abcam), anti-p27 (ab32034; Abcam), and anti-α-tubulin (ab7291; Abcam) acted as a loading controls followed by incubation with horseradish peroxidase-linked secondary antibodies (Abcam). The proteins were detected by ECL reagents (Pierce, Rockford, IL, USA).
10
Rab3D Protein Expression Analysis
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Total protein was extracted from RCC cells using RIPA Lysis (Beyotime Biotechnology, Shanghai, China) and then the protein concentration was quantified using bicinchoninic acid method (Beyotime). Then equal amounts of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and the following western blot analysis as previously described13 (link). The primary antibodies against Ras-related protein Rab-3D (Rab3D) and β-actin, as well the horseradish peroxidase-linked secondary antibody were obtained from Abcam (Cambridge, MA, USA). The protein bands were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).
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