Western Blot analysis of protein expression was perform exactly as described by us recently [44 (link)]. 20 µg of protein was used. As primary antibodies we used monoclonal mouse-anti MnSOD, catalase (Cell Signaling, Frankfurt am Main, Germany) and GAPDH (Novus, Cambridge, Great Britain) antibodies (1:1000). After incubation, membranes were washed 3 × 5 min in TBS-T (0.1% Tween 20) and incubated with secondary antibody in 5 % TBS-T (1:1000) for one hour at room temperature. As secondary antibodies we used HRP-conjugated polyclonal goat-anti-mouse antibodies (Dako, Carpinteria, CA, USA). After washing (3 × 5 min in TBS-T), membranes were incubated in enhanced luminescence reagents (Thermo Fisher Scientific). Blots were analyzed with ImageLab software (Bio-Rad). Specific protein signals were normalized to the respective GAPDH signals or to the signal of total proteins detected on the respective lanes of the gel prior blotting.
Enhanced luminescence reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced luminescence reagents are laboratory products designed to facilitate luminescence-based detection and quantification in various analytical applications. These reagents are formulated to enhance the intensity and stability of luminescent signals, enabling sensitive and reliable measurements. Their core function is to provide a reliable and sensitive method for detecting and quantifying analytes through luminescence-based assays, without making claims about specific intended uses.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Novus Biologicals (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Proteinase inhibitor mixture, by Merck Group (1 mentions)
Insulin, by Cell Signaling Technology (1 mentions)
Lamin b1, by Proteintech (1 mentions)
β tubulin, by Proteintech (1 mentions)
Hrp conjugated secondary antibody, by Sungene Biotech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
2 protocols using enhanced luminescence reagents
1
Western Blot Analysis of Antioxidant Proteins
2
Immunoblotting Analysis of Cellular Proteins
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Whole-cell lysates prepared using cold RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing a proteinase inhibitor mixture (Sigma) were quantitated using the Bradford assay (Bio-Rad). The protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against EI24 (Sigma), Insulin (Cell Signaling Technology), MAFA (Cell Signaling Technology), NOX4 (Novus Biologicals), ERO1α (R&D Systems), PRDX4 (R&D Systems), GPX7 (ABclonal), GPX8 (Novus Biologicals), RTRAF (Proteintech), Lamin B1 (Proteintech), β-tubulin (Proteintech), and β-actin (Cell Signaling Technology), followed by incubation with the appropriate HRP conjugated secondary antibodies (Sungene Biotech, Tianjin, China). Protein expression was detected using enhanced luminescence reagents (Thermo Scientific, Waltham, MA, USA).
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