Total protein was extracted using RIPA buffer (Beyotime, China), separated by 10% SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA). Then, the blots were incubated with the primary antibodies against DRP1 (1:1,000, ab184247; Abcam), LC3 (1:1,000, ab48394; Abcam), mitofusin-1 (MFN1, 1:800, 13798-1-AP; Proteintech), MFN2 (1:2,000, 12186-1-AP; Proteintech), optic atrophy-1 (OPA1, 1:1,000, DF8575; Affinity, Cincinnati, OH), TFAM (1:1,000, AF0531; Affinity), PTEN induced kinase 1 (PINK1, 1:1,000, DF7742; Affinity), mitochondrial import receptor subunit TOM20 homolog (TOMM20, 1:5,000, 11802-1-AP; Proteintech), ATP synthase subunit α (ATP5A, 1:2,000, 14676-1-AP; Proteintech), GAPDH (1:7,500, #21612; Signalway Antibody LCC, Greenbelt, MD), and tubulin (1:2,500, AF7011; Affinity) at 4°C overnight and horseradish peroxidase (HRP)-conjugated secondary antibody (ab6721, 1:15,000; Abcam). Signals were detected by ECL and quantified by a Multi Tanon 5200 imaging system. The tubulin or GAPDH was used as internal control. Protein levels were expressed as relative to Tubulin or GAPDH. The LC3-II:LC3-I ratio was calculated based on densitometry analysis of both bands.
Df7742
Manufactured by Affinity Biosciences
Sourced in China
The DF7742 is a piece of laboratory equipment produced by Affinity Biosciences. It is a centrifuge designed for the separation and purification of biological samples. The core function of the DF7742 is to facilitate the separation of different components within a sample through the application of centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Ab184247, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Af7011, by Affinity Biosciences (1 mentions)
Af0531, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab156034, by Abcam (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Phosphatase inhibitor cocktail 2, by Abcam (1 mentions)
Anti pink1, by Affinity Biosciences (1 mentions)
Anti sqstm1 p62, by Affinity Biosciences (1 mentions)
Af5384, by Affinity Biosciences (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Ab224722, by Abcam (1 mentions)
Ab219620, by Abcam (1 mentions)
3 protocols using df7742
1
Western Blot Analysis of Mitochondrial Proteins
2
Cardiac Protein Extraction and Western Blot Analysis
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Th total protein extracts were prepared from 10 to 15 mg of the deep-frozen heart tissue. To maintain the extract integrity and function, we used Complete Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), Phosphatase Inhibitor Cocktail II (ab201113, Abcam, Waltham, MA, USA), PMSF (1 mM), EGTA (1 mM), and EDTA (1 mM). The proteins were isolated using a 1xRIPA buffer (ab156034, Abcam, Waltham, MA, USA). The concentration of the protein was determined by the Lowry method [15 (link)]. The samples (30–50 µg) were diluted in Laemmli buffer, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Wilmington, USA) [12 (link)]. The relative levels of the detected proteins were visualized using a C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, Nebraska, USA) and normalized to the appropriate loading control. The antibodies used: (ab56788) anti-DRP1 (1:1000), (ab119685) anti-OPA (1:1000), (ab124773) anti-Mitofusin2 (1:1000), (ab54481) anti-PGC1a (1:1000), (cs2132) anti-Parkin (1:1000), (Affinity Biosciences, DF7742) anti-PINK1 (1:250), (Affinity Biosciences, AF5384) anti-SQSTM1/p62 (1:250), (Affinity Biosciences, DF8163) anti-BNIP3L (1:250), and (cs12741) anti-LC3A/B (1:1500). The anti-GAPDH (ab181602) and anti-tubulin (1:2000) (ab4074) antibodies were used as a loading control.
3
Safranin O-based Histological Assessment of IVDD
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Safranin O-fast green (SO) staining was performed to assess the degree of IVDD in each group. The average gray value (staining intensity) was used to grade the SO results, High positive (gray value181-236), Positive (gray value121-180), Low Positive (gray value61-120) and Negative (gray value0-60). And we grade each group as: Control group (High positive), Injured group (Low Positive), T-MN group (Low Positive), T-MN@EXO group (Positive), and T-MN@EXO@miR-378group (Positive). Col I and Fundc1 were used as immunohistochemical indicators of the degree of IVDD. The samples were treated with H2O2 (3%) for 10 min, blocked with 5% BSA for 30 min at room temperature, and hybridized with Col I antibody (ab254360, 1:100; Affinity, China) or Fundc1 antibody (ab224722, 1:100; Abcam) or MMP13 antibody (ab219620, 1:100; Abcam) or Parkin (AF0235, 1:100; Affinity, China) or PINK1 (DF7742 1:100; Affinity, China) at 4 °C overnight. The samples were then washed five times with PBS and incubated with a biotin-labeled secondary antibody at 37 °C for 30 min. The SABC method was used to detect staining.
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