Anti-SOX9 抗体 [EPR14335] (ab185230)

Western blot: Anti-Sox9 antibody [EPR14335] (ab185230) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab185230 was shown to bind specifically to Sox9. A band was observed at 50-70 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in Sox9 CRISPR-Cas9 edited cell line. The band observed in the CRISPR-Cas9 edited lysate lane below 50-70 kDa is likely to represent a truncated form of Sox9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Sox9 CRISPR-Cas9 edited HCT 116 cell lysates were analysed. 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 then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

SOX9 can be ubiquitinated or SUMOylated to higher molecular weight (PMID: 24155239, PMID: 16307912, PMID: 16554309, PMID: 32070068). Meanwhile, it has a truncated version as mini-Sox9 (PMID: 21297661,PMID: 27429045).

HeLa expresses very low level of SOX9 (PMID: 18296708, PMID: 18677406).

Immunofluorescence staining of SOX9 using ab185230 in primary hippocampal mouse neurons/glia, (obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP), DIV14. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% TritonX-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab185230 at 5 μg/ml, ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) at 1/1000 dilution. Cells were then incubated with ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green), ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple), all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected, most GFAP positive cells are also SOX9 positive, while NeuN positive cells are SOX9 negative.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunofluorescent analysis of SW480 cells (4% paraformaldehyd-fixed) labeling SOX9 with ab185230 at 1/250 dilution followed by Goat anti rabbit IgG (Alexa Fluor^® 555) secondary at 1/200 dilution and counter-stained with DAPI (blue).

Alexa Fluor^® 488 (ab196450) and Alexa Fluor^® 647 (ab196184) conjugated versions are available for this clone.

ab185230 at 1/60 immunoprecipitating SOX9 in SW480 (human colorectal adenocarcinoma) whole cell lysate observed at 56 and 75 KDa (lanes 1 and 2).

The expression pattern observed is consistent with the literature (PMID: 10682837) with an additional off-target band at 46 kD.

Lane 1 (input): SW480 whole cell lysate 10μg

Lane 2 (+): SW480 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab185230 in SW480 whole cell lysate

For western blotting, ab185230 (1/1000) was used as the primary antibody and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.

Intracellular flow cytometric analysis of PC3 cells (2% paraformaldehyde-fixed)labeling SOX9 with ab185230 at 1/190 dilution (red) or a rabbit IgG (negative) (green), followed by Goat anti rabbit IgG (FITC) secondary at 1/150 dilution. 

Alexa Fluor^®488 (ab196450) and Alexa Fluor^®647 (ab196184) conjugated versions are available for this clone. 

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.