Nucleus pulposus cells and raw264.7 were cocultured with hydrogel scaffold for 48 h, and the protein expression level was detected by WB. Total protein extraction kit (Yeasen, Shanghai, China) was used to extract cell protein. After the concentration was determined by BCA protein analysis kit (Yeasen, Shanghai, China), the protein was separated by SDS-PAGE electrophoresis and transferred to PVDF. The PVDF membrane was blocked with 5% bovine serum albumin for 1 h, and the first antibody was incubated at 4 °C. The antibodies included anti-Collagen II (Proteintech, Wuhan, China), anti-Aggrecan (Abcam, Cambridge, UK), anti-MMP13 (Proteintech, China), and GAPDH (Proteintech, Wuhan, China). The blots were produced with an HRP-conjugated secondary antibody and imaged with a ChemiDoc MP Imaging System (Bio-rad, Hercules, CA, USA). ImageJ 2.3.0 was used to measure the protein band strength and normalize it to the equivalent GAPDH bands (NIH, Bethesda, MD, USA).
Anti aggrecan
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-Aggrecan is a laboratory reagent used in research applications. It is an antibody that specifically binds to the aggrecan protein, which is a key component of the extracellular matrix in cartilage and other connective tissues. The primary function of Anti-Aggrecan is to enable the detection and quantification of aggrecan in various biological samples and experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti collagen 2, by Abcam (8 mentions)
Anti sox9, by Abcam (4 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti β actin, by Abcam (2 mentions)
Anti col2a1, by Abcam (2 mentions)
Anti adamts4, by Abcam (2 mentions)
Anti p65, by Abcam (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti adamts5, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti cd24, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Anti mmp13, by Abcam (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti collagen 2, by Proteintech (1 mentions)
Anti mmp13, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
19 protocols using anti aggrecan
1
Coculture of Nucleus Pulposus Cells and Raw264.7 in Hydrogel Scaffold
2
Chondrocyte Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reagent RIPA Lysis Buffer (Thermo Fisher Scientific) was used to extract the total protein of the cultivated chondrocyte or isolated cartilage tissue. RIPA was supplemented with protease inhibitor PMSF. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). Protein was mixed with protein loading buffer (Thermo Fisher Scientific) and loaded to a 12% SDS-PAGE gel for electrophoresis. Afterward, the gel was transferred to PVDF membrane (Thermo Fisher Scientific). After a blocking step with a blocking buffer, the membrane was incubated with primary monoclonal antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody. Finally, signal was detected by an ECL method. The primary antibodies used in this study includes anti-Aggrecan (1:1000, Abcam), anti-Collagen II (1:1000, Abcam), anti-β-actin (1:3000, Abcam), anti-ADAM8 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:2000, Abcam), anti-p- NF-κB p65 (1:1000, Abcam), anti-NF-κB p65 (1:2000, Abcam), anti-MMP9 (1:1000, Abcam), anti-Notch1 (1:1000, Abcam), anti-Hes1 (1:2000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at RT.
3
Histological Analysis of Cartilage Formation
4
Protein Expression Analysis in MBMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from each group of MBMSCs on days 3, 7, and 14 using the RIPA tissue/cell lysis buffer (Solarbio). Protein concentration was determined with a BCA kit (Beyotime, Shanghai, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V for 30 min followed at 120 V for 1 h, then transferred to polyvinylidene difluoride membranes (Millipore, USA), and blocked with Western blocking solution (Beyotime). The membrane was incubated with primary antibodies (anti-SOX9, 1 : 1000 dilution; anti-Col2A1, 1 : 1000 dilution; anti-Aggrecan, 1 : 1000 dilution; and anti-β-actin, 1 : 10,000 dilution; Abcam, USA) at 4°C overnight, followed by incubation with secondary antibodies (HRP Goat Anti-Rabbit IgG, 1 : 2000 dilution; Abcam) for 1 h. The ECL solution (Beyotime) was used for visualization and the Bio-Rad ChemiDoc™ XRS system for imaging, and the signal was captured and analyzed using Image Lab™ software (Bio-Rad).
5
Western Blot Analysis of Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Control and treated chondrocytes were lysed, followed by SDS-PAGE electrophoresis as previously described [13 ]. The primary antibodies used were listed: anti-iNOS, anti-COX-2, anti-β-actin, and HRP-conjugated secondary antibody from Santa Cruz Biotechnology, Santa Cruz, CA; and anti-ADAMTS-4, anti-ADAMTS-5, anti-aggrecan, anti-collagen II, anti-p65, anti-p-p65, anti-p-IκBα, and anti-IκBα from Abcam. Finally, the bands were visualized with the ECL reagent.
6
Intervertebral Disc Extracellular Matrix Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rats were killed as described above and the tails were obtained. The specimens of the whole IVD with adjacent vertebral bodies were harvested and cut into 5 μm with a freezing microtome (Leica, Wetzlar, Germany) at 8 weeks after injection. After being fixed in 4% paraformaldehyde for 30 min, washed twice with PBS, and blocked in 5% bull serum albumin for 1 h, the tissue sections were incubated, respectively, overnight with primary antibodies at 4°C: rabbit polyclonal anti-collagen type II and anti-aggrecan (1 : 100, Abcam, Cambridge, UK). After washing with PBS, slides were incubated, respectively, with FITC-conjugated and Cy3-conjugated goat anti-rabbit IgG (1 : 10000, ABclonal, Wuhan, China) secondary antibodies for 2 h at room temperature in the dark. The immunostaining results were photographed by a fluorescence microscope and analyzed by Image-Pro Plus 6.0 software.
7
Western Blotting Protocol for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protocol and procedure for western blotting were as described in our previous reports 30 (link), 44 (link), 45 (link). The anti-p-YAP (Ser127), anti-YAP, anti-RalA and anti-β-actin primary antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA). The anti-Wnt5a, anti-Wnt5b, anti-Aggrecan and anti-SOX9 primary antibodies were obtained from Abcam (Cambridge, MA, USA). The anti-CD63, anti-CD9, anti-CD81 and anti-Alix were obtained from System Biosciences (Palo Alto, CA, USA). Based on previous research 46 (link), 47 (link), collagen type II was analysed using 5% (wt/vol) SDS-PAGE with anti-collagen type II primary antibodies (Chemicon®, Merck-Millipore).
8
Curcumin Protects ADMSCs from Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Curcumin, bovine serum albumin (BSA), TBHP, and DMSO were obtained from sigma Sigma-Aldrich (St. Louis, MO, USA). ADMSCs were cultured in the serum-free ncMission hMSC Medium (RP02010, Nuwacell Biotechnologies Co., Ltd, China). Fetal bovine serum (FBS), collagenase II, 0.25% trypsin, 100 U/mL penicillin G and 0.1 mg/mL streptomycin were purchased from Gibco (NY, USA). Chondrocyte growth medium Dulbecco’s Modified Eagle Medium F-12 (DMEM/F12) and phosphate buffered saline (PBS) were obtained from Hyclone (Logan, UT, USA). sEV lipid fluorescent dyes DiI was purchased from Thermo Fisher Scientific (MA, USA). Annexin-V-FITC Apoptosis Detection Kit, 4′,6-diamidino-2-phenylindole (DAPI), EdU Cell Proliferation Kit with Alexa Fluor 488, and One Step TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Kit were purchased from Beyotime Biotechnology (Jiangsu, China). The following monoclone antibodies and secondary antibody were obtained from Abcam (Cambridge, UK): anti-CD63, anti-tsg101, anti-GM130, anti-8-OHdG, anti-collagen II and anti-aggrecan. The primary antibody of cleaved caspase3 was obtained from Cell Signaling Technology (Danvers, MA, USA).
9
Protein Expression Analysis in Cartilage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using RIPA buffer containing protease inhibitors. Protein samples were separated using SDS-PAGE and immunoblotting using the following primary antibodies: anti-cxcl 8 (1:1000; Abcam), anti-aggrecan (1:2000), anti-sox9 (1:2000), anti-collagen II (1:5000), and anti-RPL4 (1:1000). Then, the membranes were incubated with secondary antibodies (1:1000; all from Cell Signaling Technology). The protein bands were visualized using enhanced chemiluminescence detection kit (Millipore).
10
Comprehensive Western Blot Analysis of Cartilage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blot analysis, cells were lysed in mammalian protein extraction reagent (Pierce, Rockford, IL, USA) with complete protease inhibitor. Total protein quantity was determined using the Pierce BCA Protein Assay Kit (Pierce) and adjusted to 10 mg/mL. Samples (10 mL) were loaded onto 10–14% SDS-PAGE gels and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% skim milk in 0.05% Tris-buffered saline/Tween 20 (TBST) followed by incubation with the following primary antibodies: anti-sox9 (Abcam, Cambridge, England, 1∶1000), anti-aggrecan (Abcam, 1∶100), anti-collagen type I (Merck, Whitehouse Station, NJ, USA, 1∶500), anti-collagen type II (Merck, 1∶500) and anti-GAPDH (Cell Signaling, Danvers, MA, USA, 1∶1000), respectively. The blots were developed for chemiluminescence using the ECL western blotting substrate (Pierce).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!