Proteins were extracted from the cultured cardiomyocytes and heart tissue using RIPA buffer (Fudebio, Hangzhou, China) mixed with protease inhibitor (Fudebio, Hangzhou, China) (1:100) and quantified by BCA assay (Thermo Fisher, USA). The primary antibodies used were anti-β-MHC (1:1000, Abcam, USA), anti-ANP (1:500, Abcam, USA), anti-TGF-β1 (1:1000, Proteintech, USA), anti-PTEN-induced putative kinase-1 (PINK1) (1:1000, Santa Cruz, USA), anti-Parkin (1:1000, Abcam, USA), anti-caspase-3 (1:1000, Cell Signalling Technology, USA), anti-Bcl-2 (1:1000, Proteintech, USA), and anti-β-actin (1:3000, Proteintech, USA). The secondary antibodies used were goat anti-rabbit and anti-mouse IgG-HRP (1:3000, Fudebio, Hangzhou, China). Bands were detected with ECL substrate (Fudebio, Hangzhou, China) and visualized with a GeneGnome imaging system (Syngene Bioimaging). Densitometry was carried out with ImageJ (NIH).
Protease inhibitor
Manufactured by Fudebio
Sourced in China
Protease inhibitor is a lab equipment product used to inhibit the activity of proteases, enzymes that break down proteins. It functions by binding to and blocking the active site of proteases, preventing them from cleaving their target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β mhc, by Abcam (1 mentions)
Anti parkin, by Abcam (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti anp, by Abcam (1 mentions)
Ecl substrate, by Fudebio (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti tgf β1, by Proteintech (1 mentions)
Genegnome imaging system, by Syngene (1 mentions)
Ripa buffer, by Fudebio (1 mentions)
Anti β tubulin, by Abcam (1 mentions)
Ab34712, by Proteintech (1 mentions)
Ab39012, by Proteintech (1 mentions)
Ab205606, by Proteintech (1 mentions)
C8035, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
5 protocols using protease inhibitor
1
Protein Expression Analysis in Cardiomyocytes
2
Western Blot Protein Extraction Protocol
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Total protein was extracted from cells and tissues using RIPA lysis buffer containing protease inhibitor (Fudebio, Hangzhou, China). Protein lysates were separated by SDS-PAGE gels and subsequently electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Then the membranes were blocked for 1 h with 5% nonfat skim milk in TBST and incubated with specific primary antibodies at 4 °C overnight. Then, the membranes were washed with TBST and incubated with a secondary antibody for 1 h (Fudebio, Hangzhou, China). Anti-beta tubulin were purchased from Abcam (Cambridge, UK) and anti-HNRNPA1 antibodies were purchased from ABclonal (Wuhan, China).
3
Western Blot Analysis of Chondrocyte Markers
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Protein extraction was performed by lysing primary MCs and C28/I2 cells in RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor (Fudebio, Hangzhou, China). The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime). Equivalent amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore). The membranes were blocked and then incubated with primary antibodies for 12 h at 4°C, then incubated with corresponding secondary antibodies (Proteintech, Wuhan, China) for one hour. β‐actin was used as the internal standard, and the relative grey level of proteins was calculated using ImageJ software (NIH, Bethesda, MD, USA). The antibodies used were as follows: anti‐β‐actin antibody (1:2000, Proteintech, 66009‐1‐Ig), anti‐LOX1 antibody (1:1000, Proteintech, 11837‐1‐AP), anti‐Aggrecan antibody (1:1000, Sigma–Aldrich, c8035), anti‐COL2A1 antibody (1:1000, abcam, ab34712), anti‐MMP3 (1:1000, Proteintech, 17873‐1‐AP), anti‐MMP13 (1:1000, abcam, ab39012), anti‐ADAMTS4 (1:1000, Proteintech, 11865‐1AP), anti‐ADAMTS5 (1:1000, abcam, ab41037), anti‐Vimentin (1:1000, Cell Signaling Technology, 5741), anti‐FLAG (1:1000, abcam, ab205606), and anti‐SYVN1 (1:1000, Proteintech, 67488‐1‐Ig).
4
Immunoprecipitation of ASC Protein
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Cells were lysed in a RIPA buffer containing (50 mM Tris(pH7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitors (Fudebio), phosphatase inhibitors (Fudebio)). After centrifugation at 20,000 x g for 10 min, the supernatant was incubated with either IgG control antibody (sc-3877, Santacruz) or anti-ASC antibody (67494-1-Ig, Proteintech) with protein A/G PLUS-Agarose (Santacruz) overnight at 4 °C. After washing with the above lysis buffer, the immunoprecipitated proteins were collected by centrifugation at 20,000 x g for 10 min, resuspended and boiled in 1× SDS loading buffer at 95 °C for 10 min.
5
Western Blot Analysis of Protein Expression
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Total protein from homogenized tissues or cell lysates was extracted using ice-cold RIPA solution (Fudebio, China) and protease inhibitors (Fudebio, China). The protein samples were diluted to equal concentrations, denatured in a boiling water bath, separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline-Tween (TBST) buffer and were then incubated overnight at 4 °C with primary antibodies against cleaved CASP3 (1:1000) (Affinity Biosciences, USA), CCND1 (1:1200) (Proteintech, China), CDK4 (1:2000) (Abcam, USA), BAX (1:1000) (Abcam, USA), BCL2 (1:800) (Abcam, USA), E-cadherin (1:2000) (Proteintech, China), N-cadherin (1:2000) (Proteintech, China), Vimentin (1:3000) (Proteintech, China), FGF2 (1:200) (Santa Cruz Biotechnology, USA), PI3K (1:1000) (Cell Signaling Technology, USA), phosphorylated PI3K (p-PI3K, 1:1000) (Cell Signaling Technology, USA), Akt (1:1000) (Cell Signaling Technology, USA), phosphorylated Akt (p-Akt, 1:1000) (Cell Signaling Technology, USA) and GAPDH (1:10000) (Proteintech, China). After four washes with TBST buffer, membranes were incubated for 60 min at 25 °C with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000) (Bioss, China). Protein bands were visualized with a chemiluminescence imaging system (Bio-Rad, USA).
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