Total protein was isolated using RIPA lysis buffer (Applygen Technologies Inc). BCA assay (Thermo Fisher Scientific, Inc) was used to determine the protein levels. The protein lysate was then separated through SDS‐PAGE (Beijing Biosynthesis Biotechnology) and transferred onto PDVF membrane. After blocking with skim milk (Solarbio Inc) for 1 hours at room temperature, the membrane was added with anti‐Smad3 (1:2000 dilution; Abcam), anti‐RUNX2 (1:2000 dilution; Abcam), anti‐collagen type X (1:2000 dilution; Abcam), anti‐Indian Hedgehog (1:2000 dilution; Abcam), anti‐OPN (1:2000 dilution; Abcam) and anti‐OCN (1:2000 dilution; Abcam) primary antibodies. After overnight incubation at 4°C, the membrane was rinsed in 1 × TBST (Solarbio Inc) and then incubated with HRP‐labelled rabbit antimouse (1:3000 dilution; Abcam, USA) secondary antibody at room temperature for 1 hours. We employed GAPDH as the internal control (1:3000, Abcam). Additionally, when > 1 protein was presented, the Western blotting membranes were stripped and reprobed with WB Stripping buffer (No: 21059; Thermo Fisher Scientific, USA) and different primary antibodies, respectively. The grey values were measured by Image Lab (v 5.2.1).
Anti opn
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-OPN is a monoclonal antibody that specifically recognizes osteopontin (OPN). OPN is a secreted, multifunctional glycoprotein involved in various biological processes such as bone mineralization, immune cell function, and tissue repair.
Automatically generated - may contain errors
Lab products found in correlation
Anti runx2, by Abcam (14 mentions)
Anti ocn, by Abcam (10 mentions)
Anti β actin, by Abcam (7 mentions)
Anti bmp2, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti alp, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Anti osterix, by Abcam (3 mentions)
Anti lef1, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Anti col1a1, by Abcam (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti col1, by Abcam (3 mentions)
Gapdh, by Abcam (2 mentions)
Anti vimentin, by Abcam (2 mentions)
Confocal laser scanning microscope, by Zeiss (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (2 mentions)
Anti ank, by Abcam (2 mentions)
48 protocols using anti opn
1
Evaluation of Osteogenic Markers in Cells
2
Evaluating EMT Markers by Western Blot
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The protein expression levels of OPN, Vimentin, E-cadherin, N-cadherin, Twist1, AXL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were evaluated by Western blot. Total protein was extracted from cells by lysing the cells in RIPA buffer [50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% NP-40, 0.25% Na-deoxydiolate, 1 mM EDTA] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 mM Na3VO4, 1 Mm NaF). Protein samples was separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene fluoride) membranes. After blocked with 5% non-fat milk/TBST, the membrane was incubated with the primary antibody. The following primary antibodies were used: anti-OPN (Abcam, Cambridge, UK), anti-Vimentin (Abcam), anti E-cadherin (Cell Signal Tech, Danvers, USA), anti-N-cadherin (Millipore, Massachusetts, USA), anti-Twist1 (Abcam), anti-Snail (Cell Signal Tech), anti-AXL (Abcam), or anti-GAPDH (Cell Signal Tech); and detected with enhanced chemiluminescence reagents (Thermo Fisher Scientific). Bands were acquired by Molecular Imager ChemiDox XRS+ Imaging System with Quantity One Image software (Bio-Rad Laboratories).
3
Immunofluorescence Staining of OPN
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Transfected cells grown on glass coverslips were fixed for 30 min in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 (Promega) in PBS buffered saline for 30 min. After washing with TBS-0.1% Triton X-100 (TBSTx), nonspecific binding sites were blocked with TBSTx-5% BSA (Roche) for 60 min. Then cells were applied sequentially with a 1:100 dilution of rabbit polyclonal antibody anti-OPN (Abcam) at 4°C overnight and a 1:200 dilution of Cy3-conjugated goat anti-rabbit IgG (Beyotime) at room temperature for 60 min in the dark. For negative control, PBS was added instead of primary antibody. Further, immunofluorescence was visualized under a laser-scanning confocal microscope (Zeiss, Oberkochen, Germany).
4
Immunohistochemical Analysis of OPN and OC
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Sections were deparaffinized with xylenes and rehydrated in a descending series of ethanol concentrations. Citrate buffer (10 mM, pH 6.0) was used to do antigen retrieval at 95 °C for 25 min. After antigen retrieval, sections were allowed to cool down to room temperature. Sections were blocked for endogenous peroxidase activity with 0.3% hydrogen peroxide for 20 min. Nonspecific protein binding was blocked by incubation with 2% bovine serum albumin and then incubated with the anti-OPN (Abcam, USA; diluted 1:300) or anti-OC (Bioss, China; diluted 1:50) overnight at 4 °C in a humidified chamber. The secondary antibody was applied, carried horseradish peroxidase and developed according to the manufacturer’s protocol (IHC staining module, Beijing Zhongshan Biotechnology, China). Three samples for each group were analyzed by an inverted light microscopy (Olympus IX71, Japan).
5
Protein Expression Analysis in Cells
6
Osteogenic Differentiation and ROS Regulation
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Annexin V-FITC/PI detection kit was purchased from BD Biosciences (San Jose, CA, USA). Fluorometric Intracellular ROS Kit and N-acetyl-L-cysteine (NAC) were from Sigma-Aldrich (St. Louis, USA). Information of primary antibodies is listed as follows: anti-Runx2 (1 : 500, Abcam), anti-Osterix (1 : 500, Abcam), anti-OPN (1 : 1000, Abcam), anti-BSP (1 : 1000, CST), anti-OCN (1 : 500, Abcam), anti-NADPH oxidase 4 (NOX4) (1 : 500, Abcam), anti-SOD2 (1 : 500, Abcam), anti-Caspase-3 (1 : 1000, CST), and anti-GAPDH (1 : 5000, Bioworld Technology Inc., USA). ALP staining and alizarin red were both from Cyagen (Guangzhou, China). For semiquantitative analysis, p-nitrophenyl phosphate (p-NPP) and 10% cetylpyridinium chloride were from Sigma-Aldrich (St. Louis, USA). Alexa Fluor 488 conjugated was from Jackson ImmunoResearch (USA). DAPI was from Sigma-Aldrich (St. Louis, USA). For scaffold fabrication, gelatin, carboxymethyl chitosan, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were all purchased from Aladdin (Shanghai, China). Calcein AM (4 mM) and Lipofectamine 3000 were from Thermo Fisher Scientific (USA).
7
Protein Expression Analysis by Western Blot
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Western blot was conducted according to a previously reported study35 (link). Total cell proteins were extracted using protein lysis buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Total cell protein concentration was measured using a BCA kit (Pierce, Rockford, IL, USA), then equal amount of proteins were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), separated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membrane was then incubated with the following primary antibodies: anti-STAT3, anti-phospho-STAT3 (Cell Signaling Technology, Inc. Danvers, MA, USA), anti-OPN, anti-Runx2, anti-Osterix, anti-SMAD4, anti-JMJD3 and anti-β-actin (All from Abcam, Cambridge, MA, USA). Then the membrane was incubated with DylightTM 800 secondary antibody (Invitrogen), the immunoblots were imaged by LI-COR Odyssey infrared Imaging System (LI-COR, Lincoln, NE, USA).
8
Osteogenic Differentiation of BMSCs
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Third‐passage BMSCs were incubated in a 20‐cm glass bottom cell culture dish. After 3 days of osteogenic induction, cells were fixed, washed, blocked and incubated in anti‐RUNX2 and anti‐OPN (both 1:100, Abcam) for 12 h. The rest of the experimental procedures were performed in a dark environment. Next day, the goat antibody (1:200, Beyotime) was incubated with the cells for 1 h. The cytoskeleton was stained with phalloidin (1:100, cytoskeleton, America) for 30 min. The nuclei were stained by 4′6‐diamidino‐2‐phenylindole (DAPI, Beyotime, China) for 15 min, and the images were captured by microscope (Leica).
9
Immunohistochemical Analysis of Bone Markers
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The slides underwent dewaxing, dehydration, rehydration, antigen retrieval, and blocking. They were then incubated with primary antibodies overnight, including anti-OPN (1:300, Abcam, Cambridge, United Kingdom), anti-RUNX2 (1:300, Abcam, Cambridge, United Kingdom), and anti-ERK5 (1:300, Affinity, Melbourne, Australia), followed by incubation with secondary antibodies (anti-mouse and anti-rabbit; Servicebio, Wuhan, China). The images were captured by the microscope system (Olympus, Japan); in each slice, three images from different visions were captured. The experiment was repeated three times. The H-score was calculated for expression analysis of OPN, Runx2, and ERK5, using the following formula: H-Score = ∑(pi×i) = (percentage of weak intensity ×1) + (percentage of moderate intensity ×2) + (percentage of strong intensity ×3) (Maclean et al., 2020 (link)).
10
Protein Extraction and Western Blot Analysis
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DPCs were isolated by differential digestion as described above, after which total protein was extracted and normalized according to the manufacturer’s instructions. The primary antibodies were anti-APE1 (1:1,000; abcam, USA), anti-DMP1 (1:1,000; Santa cruz, USA), anti-DSP1-H (1:1,000; Santa cruz, USA), anti-OPN (1:1000; abcam, USA), anti-ALP (1:1,000; abcam, USA), anti-OSX (1:1,000; abcam, USA), anti-Axin (1:1000; abcam, USA), anti-Lef1 (1:1000; abcam, USA), anti-non-p (active) β-catenin (1:1000; CST, USA), anti-p-GSK-3β (1:1000; CST, USA), anti-P21 (1:1000; abcam, USA), anti-cyclin-D1 (1:1,000; Santa cruz, USA) and anti-GAPDH (1:10,000; Zen, China) used as internal control. Then, the membranes were rinsed with TBST (0.1% Tween-20 in 0.01 mol/L TBS), incubated with appropriate horseradish peroxidase conjugated secondary antibodies at 1:5000 (Santa Cruz, USA) at room temperature for additional 2 h, visualized by Image Quant LAS 4000 mini (GE, UK). Densitometry analysis on the bands was performed using the NIH image J software and normalizing the data to total protein levels (Supplementary Fig. 1 . and Supplementary Fig. 2 ).
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