The protein was extracted from 10 mg articular cartilage using a protein isolation kit (ReadyPrep; GE Healthcare Life Sciences). The protein concentration was determined using a bicinchoninic assay kit (Thermo Fisher Scientific, Inc.). The protein (20 μg) was separated on 12% SDS PAGE gel and transferred to the nitrocellulose membrane. At room temperature, the membrane was sealed in 5% skimmed milk for 2 hours and incubated with the following primary antibodies at 4°C overnight: Anti-LC3 I, Anti-LC3 II, anti-MMP13, anti-aggrecan, anti-collagen II, anti-TGFβR1, anti-β-actin (Cell Signaling Technology, Inc., Danvers, USA). The next day, the nitrocellulose membranes were washed three times and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The protein bands were detected by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) scanned by ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein expression was normalized to β-actin and densitometric analysis was performed by ImageJ Software version 7.0 (National Institutes of Health, Bethesda, MD, USA).
Readyprep
Manufactured by GE Healthcare
Sourced in United States
ReadyPrep is a laboratory equipment product designed for sample preparation. It offers a standardized and consistent approach to sample preparation, intended to facilitate subsequent analysis procedures.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti lc3 1, by Cell Signaling Technology (1 mentions)
Anti lc3 2, by Cell Signaling Technology (1 mentions)
Anti mmp13, by Cell Signaling Technology (1 mentions)
Anti aggrecan, by Cell Signaling Technology (1 mentions)
Anti collagen 2, by Cell Signaling Technology (1 mentions)
Anti tgfβr1, by Cell Signaling Technology (1 mentions)
Ab131368, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab48394, by Abcam (1 mentions)
Ta 08, by ZSGB-BIO (1 mentions)
Bs 1313r, by Bioss Antibodies (1 mentions)
Af5135, by Affinity Biosciences (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Anti vimentin, by Beyotime (1 mentions)
Anti n cadherin, by Beyotime (1 mentions)
Electrochemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
6 protocols using readyprep
1
Cartilage Protein Extraction and Analysis
2
Protein Extraction and Western Blot Analysis
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The total protein was abstracted by adding the corresponding pyrolysis solution (4°C for 30 minutes) (28–9425–44, ReadyPrep; GE Healthcare Life Sciences). After centrifugation at 876×g for 10 minutes, the supernatant was collected. Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were processed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later transferred onto nitrocellulose membrane as previously described [11 (link)]. After blocking with 5% nonfat milk (with Tween-20) at room temperature for 2 hours, the membrane were incubated with the primary antibodies against GAPDH (1:1000, TA-08, ZSbio, China), VEGF (1:1000, bs-1313R, Bioss), Sirt3 (1:1000, AF5135, Affinity), and LC3 (1:1000, ab48394, Abcam) overnight at 4°C. The secondary antibody (1:10 000, ab131368, Abcam, USA) was incubated with the membrane for 2 hours at room temperature. The expression of target proteins was normalized to GAPDH. At least 6 repeats were included in the western blotting.
3
Protein Expression Analysis of Huc-MSCs-exo
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At 24 h post-treatment with Huc-MSCs-exo, protein was extracted from cells using the triplePrep kit (cat. no. 28-9425-44; ReadyPrep; GE Healthcare Life Sciences). The protein levels were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. A total of 25 µg/lane protein was separated via SDS-PAGE on a 10% gel and transferred onto nitrocellulose membranes. The membranes were blocked using 5% skimmed milk at room temperature for 2 h and incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-E-cadherin (1:1,500; cat. no. AF0131; Affinity Biosciences), anti-GAPDH (1:1,000; cat. no. AG019; Beyotime Institute of Biotechnology), anti-Vimentin and anti-N-cadherin (1:3,000 and 1:1,000, respectively; cat. nos. ab92547 and ab76057, respectively; both Abcam). After washing with 0.1 M PBS, the membranes were incubated with the secondary antibody (HRP-labeled goat anti-rabbit IgG; cat. no. A16104; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The blots were visualized using an electrochemiluminescence kit (Thermo Fisher Scientific, Inc.). The densities of the blots were quantified using the Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.).
4
Protein Quantification and ELISA Assay Protocol
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The cells were collected and total protein was isolated using a triple prep kit (cat. no. 28-9425-44, ReadyPrep; GE Healthcare Life Sciences). Protein concentration was measured using a bicinchoninic acid assay kit (cat. no. P0009: Beyotime Institute of Biotechnology). Volumes were adjusted to normalize the protein content, and then aliquots were processed for the ELISA using rat TGF-β1 (cat. no. MM-0181R1), rat MMP-9 (cat. no. MM-20918R1) and rat PDGF assay kits (cat. no. MM-0076R1; all MlBio; Shanghai Enzyme Biotechnology Co., Ltd.).
5
Western Blot Analysis of Osteogenic Markers
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Total proteins were abstracted from the cells by adding cell lysate solution (28-9425-44, ReadyPrep, GE Healthcare Life Sciences, USA). In brief, the cells with lysate solution were put on ice for 30 min and centrifuged at 4°C for 10 min (10000 ×g), and the supernatant was discarded. The protein concentration was determined using the BCA kit. The protein was denatured and run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 1–2 h, followed by wet transferring to nitrocellulose membrane for 30–50 min. The membranes were blocked in 50 defat milk for 2 h at room temperature and incubated with the primary antibodies at 4°C overnight. The primary antibodies included rabbit polyclonal anti-ALP (1 : 1000, PA5-69994, Thermofisher, Shanghai, China), rabbit polyclonal anti-RUNX2 (1 : 200, bs-1134R, Bioss; Shanghai, China), rabbit polyclonal anti-COL-I (1 : 200, bs-0578R, Bioss, Shanghai, China), rabbit polyclonal anti-SP7 (1 : 500, bs-1110R, Bioss, Shanghai, China), and mouse monoclonal anti-OCN (1 : 500, OM266706, OmnimAbs; Shanghai, China). The membranes were then incubated with the secondary antibodies. ECL exposure solution was added to the membrane and exposed to the gel imaging system. The gray values of bands were analyzed by “Quantity One” software.
6
Western Blot Analysis of Protein Expression
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Following treatments, protein was extracted using a protein isolation kit (cat. no. 28-9425-44, ReadyPrep; GE Healthcare Life Sciences) (containing phenylmethane sulfonyl fluoride), as previously described (16 (link)). Protein samples were heated at 100°C for 10 min, and the protein concentration was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Proteins (25 µg/well) were separated by SDS-PAGE (12% gel), and protein was then transferred onto a nitrocellulose membrane. Membrane was blocked with 5% nonfat milk in phosphate-buffered saline with Tween-20 at room temperature for 2 h. Primary antibodies against, PD-L1 (1:1,000; cat. no. ab199380; Abcam), AKT (1:1,000; cat. no. bs-0115R; BIOSS), PI3K (1:1,000; cat. no. ab191606; Abcam) and GAPDH (1:1,000; cat. no. ab24071; Abcam) were incubated with membranes overnight at 4°C. After washing (3 times for 10 min each), membranes were incubated with the goat Anti-Rabbit IgG H&L (HRP) (1:10,000; cat. no. ab131368; Abcam) for 2 h at room temperature. Chemiluminescent substrate detection reagent (cat. no. RPN2133; GE Healthcare Life Sciences) was applied to reveal the signals. Target bands were analyzed by ImageJ software for grayscale analysis.
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