To analyze the protein expression, western blot analysis was performed as previously described.47 (link), 49 (link) The primary antibodies to Nrf2, HDAC1, SIRT1, SIRT6, and AMPK were purchased from Abcam (Cambridge, MA, USA). Anti-LKB1 was obtained from Sigma-Aldrich (Sigma, MO, USA), anti-HO-1, anti-NQO-1, anti-CAT, anti-IKK, anti-IκBα, anti-β-Klotho, anti-CTGF, and anti-TGF-β were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p65, anti-caspase-8, anti-caspase-3, anti-PARP, anti-Bax, anti-Bcl-2, anti-FGFR1, and anti-acetylated-lysine were purchased from Cell Signaling (Danvers, MA, USA). Anti-3-NT was from Millipore (Billerica, CA, USA), and anti-4-HNE was from Alpha Diagnostic International (San Antonio, TX, USA). After three washes with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the membrane was reacted with appropriate secondary antibodies for 1 h at room temperature. Finally, the probed proteins were stained with enhanced chemiluminescence reagent and visualized using the BIO-RAD ChemiDoc Touch Imaging System (BIO-RAD, Hercules, CA, USA).
Anti 4 hne
Manufactured by Alpha Diagnostic
Sourced in United States
Anti-4-HNE is a laboratory reagent used for the detection and quantification of 4-hydroxy-2-nonenal (4-HNE), a biomarker of oxidative stress. It is designed for use in immunoassays and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti 3nt, by Merck Group (3 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti caspase 8, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti nrf2, by Santa Cruz Biotechnology (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti histone h3, by Santa Cruz Biotechnology (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Anti cat, by Santa Cruz Biotechnology (1 mentions)
Anti acetylated lysine, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti nqo1, by Santa Cruz Biotechnology (1 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Anti fgfr1, by Cell Signaling Technology (1 mentions)
Anti ho 1, by Santa Cruz Biotechnology (1 mentions)
Anti tgf β, by Santa Cruz Biotechnology (1 mentions)
Sirt1, by Abcam (1 mentions)
Hdac1, by Abcam (1 mentions)
Sirt6, by Abcam (1 mentions)
5 protocols using anti 4 hne
1
Western Blot Analysis of Protein Expression
2
Metabolic and Protein Analysis in Skeletal Muscle
3
Western Blot Analysis of Testis Oxidative Stress Markers
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Western blot was performed using testis tissue as described in our previous study [38 (link)]. The primary antibodies included anti-3-NT (Millipore, Temecula, CA, USA; 1 : 1,000), anti-4-HNE (Alpha Diagnostic, San Antonio, TX, USA; 1 : 3,000), anti-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, 1 : 2,000), anti-ATF4 (Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti-Bax (Cell Signaling Technology, 1 : 1000), anti-Bcl-2 (Santa Cruz Biotechnology, 1 : 2,000), anti-caspase-3 (Cell Signaling Technology, 1 : 1000), anti-caspase-8 (Cell Signaling Technology, 1 : 1000), anti-caspase-12 (Cell Signaling Technology, 1 : 1000), anti-CHOP (Cell Signaling Technology, 1 : 1000), anti-Histone H3 (Santa Cruz Biotechnology; 1 : 500), anti-IL-6 (Cell Signaling Technology, 1 : 1000), anti-NRF2 (Santa Cruz Biotechnology, 1 : 1000), anti-TNF-α (Abcam, 1 : 2,000), and anti-VCAM-1 (Santa Cruz Biotechnology, 1 : 500)
4
Immunoblotting Analysis of 4-HNE Protein Adducts
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Samples from all time points were resuspended in a lysis buffer consisting of 1% (w/v)
urea, 2 M thiourea, and 40 mM Tris (pH 10.4) at a final concentration of 20 x 10 6 /100 µL and incubated for 1 h (4°C) with constant rotation. After centrifugation (18,000 xg, 15 min, 4°C), the supernatant was recovered and total protein was quantified using a 2-D quant kit (G.E.
Healthcare, Sydney, Australia) following manufacturer's protocol. Samples were then used for immunoblotting.
Immunoblotting was carried out as described elsewhere (53). Essentially, 30 g of protein was loaded into SDS-PAGE and the samples run until the lowest mass pre-stained marker reached the bottom of the gel. Transfer to nitrocellulose occurred by running the samples at 350 mA for 60 min. Proteins transferred onto the nitrocellulose membrane were blocked (3% BSA-TBST), and then incubated with the polyclonal antibody anti-4-HNE (Alpha Diagnostics, San Antonio, USA) diluted 1:1000 in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-20. The secondary goat anti-rabbit immunoglobulin G horseradish peroxidase (HRP) conjugate was used at a concentration of 1:1000 in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-20. Stripping of the blots was performed as described elsewhere, and they were blotted again with antiubulin (1:1000) as a mean of loading control [50] .
urea, 2 M thiourea, and 40 mM Tris (pH 10.4) at a final concentration of 20 x 10 6 /100 µL and incubated for 1 h (4°C) with constant rotation. After centrifugation (18,000 xg, 15 min, 4°C), the supernatant was recovered and total protein was quantified using a 2-D quant kit (G.E.
Healthcare, Sydney, Australia) following manufacturer's protocol. Samples were then used for immunoblotting.
Immunoblotting was carried out as described elsewhere (53). Essentially, 30 g of protein was loaded into SDS-PAGE and the samples run until the lowest mass pre-stained marker reached the bottom of the gel. Transfer to nitrocellulose occurred by running the samples at 350 mA for 60 min. Proteins transferred onto the nitrocellulose membrane were blocked (3% BSA-TBST), and then incubated with the polyclonal antibody anti-4-HNE (Alpha Diagnostics, San Antonio, USA) diluted 1:1000 in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-20. The secondary goat anti-rabbit immunoglobulin G horseradish peroxidase (HRP) conjugate was used at a concentration of 1:1000 in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween-20. Stripping of the blots was performed as described elsewhere, and they were blotted again with antiubulin (1:1000) as a mean of loading control [50] .
5
Comprehensive Kidney Protein Analysis
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