Recombinant Anti-SOX17 antibody [EPR20684] (ab224637)

ab224637 staining SOX17 in wild-type HeLa cells (top panel) and SOX17 knockout HeLa cells (ab265744) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes 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 with ab224637 at 0.2μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Immunohistochemical analysis of paraffin-embedded human seminoma tissue labeling SOX17 with ab224637 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining on tumor cells of human seminoma (PMID:19369635; PMID:18348160) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-SOX17 antibody [EPR20684] (ab224637) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SOX17 knockout HeLa cell lysate
Lane 3 : SK-OV-3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 44 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab224637 observed at 51 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab224637 Anti-SOX17 antibody [EPR20684] was shown to specifically react with SOX17 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265744 (knockout cell lysate ab257697) was used.  Wild-type and SOX17 knockout samples were subjected to SDS-PAGE.  ab224637 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-SOX17 antibody [EPR20684] (ab224637) at 1/500 dilution

Lane 1 : SK-OV-3 (Human adenocarcinoma) whole cell lysates with NFDM/TBST
Lane 2 : NIH: OVCAR-3 (Human ovary adenocarcinoma) whole cell lysates with NFDM/TBST
Lane 3 : HeLa (Human cervix adenocarcinoma) whole cell lysates with NFDM/TBST
Lane 4 : Rat E14 embryo tissue lysates with NFDM/TBST
Lane 5 : Rat E9.5 embryo tissue lysates with NFDM/TBST
Lane 6 : Rat lung tissue lysates with NFDM/TBST

Lysates/proteins at 20 µg per lane.

Blocking peptides at 5 % per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG) at 1/2000 dilution

Predicted band size: 44 kDa

Exposure time: 
Lanes 1-2: 5 seconds
Lanes 3-6: 3 minutes

The expression profile observed is consistent with the literature (PMID: 11786926).

Negative control: HeLa.

SOX17 was immunoprecipitated from 0.35 mg of SK-OV-3 (human ovarian cancer cell line) lysate with ab224637 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224637 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: SK-OV-3 whole cell lysate 10 µg (Input). 

Lane 2: ab224637 IP in SK-OV-3 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab224637 in SK-OV-3 whole cell lysate.

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

Exposure time: 1 second.

Immunohistochemical analysis of paraffin-embedded human choriocarcinoma tissue labeling SOX17 with ab224637 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Negative tissue: no staining on tumor cells of human choriocarcinoma (PMID: 19369635) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling SOX17 with ab224637 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining on endothelium of rat lung (PMID:24418654) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling SOX17 with ab224637 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining on endothelium of mouse spleen (PMID:24418654) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.