Recombinant Anti-SOX9 antibody [EPR14335] (ab185230)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Protein concentrations of samples were measured using bicinchoninic acid (BCA) assays, and samples containing equal volumes of lysis buffer from an equal number of similarly-sized explants or chondrocytes were loaded onto gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Cartilage and cell extract samples containing 20 µg of protein were separated by 10% Bis-Tris Gel NuPAGE® electrophoresis using 5% MOPS SDS Running Buffer (Novex, CA, USA). Separated proteins were dry blotted onto nitrocellulose iBlot®gel transfer stacks in iBlot Gel transfer devices for 7–10 min. The nitrocellulose membrane was blocked by shaking in Blocking One (Nacalai Tesque, Kyoto, Japan) for 60 min at room temperature. The blots were subsequently shaken overnight at 4 °C in solution containing the following specific primary antibodies: mouse anti-HIF-1α (1:500 dilution; Novusbio catalog no. NB100-105), rabbit anti-SOX9 (1:1000 dilution; Abcam catalog no. ab185230), rabbit anti-HABP2 (1:10,000 dilution; Abcam catalog no. ab181837), and rabbit anti-CD44 (1:200 dilution; Abcam catalog no. ab24504), and with mouse anti-β-actin (1:4000 dilution; Sigma-Aldrich catalog no. A2228) as loading controls. Blots were washed three times with Tris Buffered Saline with Tween 20 (TBST) and shaken for 60 min at room temperature with peroxidase-conjugated anti-mouse IgG (1:4000 dilution; Sigma-Aldrich catalog no. A4416) or peroxidase-conjugated anti-rabbit IgG (1:4000 dilution; Sigma-Aldrich catalog no. A0545) secondary antibodies, as appropriate. The blots were again washed three times with TBST, and chemiluminescence emission was visualized using Chemi-Lumi One L (Nacalai Tesque). Protein band intensities were assessed using an ECL Select LAS500 (GE Healthcare). Band densities were quantified using Image J software (National Institute of Health, Bethesda, MD, USA), and presented as relative level to β-actin which is the internal control. The same assay as above was conducted in two independent experiments.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-SOX9 antibody [EPR14335] (ab185230) below.

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