Western blot - Anti-4 Hydroxynonenal antibody (ab46545)

Frozen mouse cardiac tissue was homogenized with lysis buffer containing 50 mmol/L Tris-HCl (pH7.5), 5 mmol/L EDTA, 10 mmol/L EGTA, 1X cock tail protease inhibitor, 1X alkaline phosphatase inhibitor and 1X acid phophatase inhibitor, 50 ug/ml phenylmenthysulfonyl fluoride and 1.23 mg/ml Chaps. Extracts were centrifuged at 12,000 rpm at 4°C for 15 minutes. 10 ug of the sample proteins was mixed with loading buffer (40 mmol/L Tris-HCl, pH 6.8, 1% SDS, 50 mmol/L DTT, 7.5% glycerol and 0.003% bromophenol blue and heated at 95°C for 5 minutes, and subjected to electrophoresis on a gradient gel (4% to 12%) at 120V. After electrophoresis, the protein was transferred to a PVDF membrane in a transfer buffer. The PVDF membrane was rinsed briefly in TBS buffer containing 50 mM Tris, 137 mM NaCl, pH 7.5 and blocked in buffer (5% milk with 0.5% BSA in TBST buffer (TBS buffer containing 0.1% tween 20) at room temperature for 1 hour. The membrane was then incubated with rabbit anti 4-hydroxy-2-noneal (4HNE) antibody at 1/3000 dilution at 4°C over night, followed by washing three times. The secondary antibody was incubated with the membrane for another one hour at room temperature. Finally the antigen-antibody complexes were visualized with use of an enhanced chemiluminescence kit. Anti-GAPDH (Abcam) was used for normalizing.

All lanes:

Western blot - Anti-4 Hydroxynonenal antibody (ab46545)

Image from Wang J et al., PLoS One. 2013;8(1):e53951. Fig 7(A).; doi: 10.1371/journal.pone.0053951. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

Western blot - Anti-4 Hydroxynonenal antibody (ab46545)

Western blot: Anti-4-HNE antibody (ab46545) staining at 1/1000 dilution, shown in black. In Western blot, ab46545 binds to 4-HNE but shows some non-specific binding to BSA. We recommend ab48506 for Western blot of 4-HNE. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with a high-sensitivity ECL substrate kit and imaged with 3 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) at 1/50000 dilution.

Lanes 1 - 4:

Western blot - Anti-4 Hydroxynonenal antibody (ab46545) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-4 Hydroxynonenal antibody [HNEJ-2] (ab48506) at 1/1000 dilution

Lane 1:

BSA cell lysate at 0.5 µg

Lane 2:

BSA cell lysate at 1 µg

Lane 3:

4-Hydroxynonenal (BSA) cell lysate at 0.5 µg

Lane 4:

4-Hydroxynonenal (BSA) cell lysate at 1 µg

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 66 kDa