All lanes : Anti-DAB2 antibody [EPR22881-69] (ab256524) at 1/1000 dilution

Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : DAB2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : Caco-2 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

Observed band size: 95 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab256524 observed at 95 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab256524 Anti-DAB2 antibody [EPR22881-69]  was shown to specifically react with DAB2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265731 (knockout cell lysate ab257910) was used. Wild-type and DAB2 knockout samples were subjected to SDS-PAGE. ab256524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling DAB2 with ab256524 at 1/500 dilution (0.14 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in rat kidney (PMID: 30052485) is observed. The section was incubated with ab256524 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

All lanes : Anti-DAB2 antibody [EPR22881-69] (ab256524) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : Caco-2 (human colorectal adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 95 kDa why is the actual band size different from the predicted?

The actual band size higher than predicted is mainly caused by phosphorylation (PMID: 28876503). 

The molecular weight observed is consistent with what has been described in the literature (PMID: 24648493).

Exposure time: Lane 1: 5.5 seconds Lane 2: 15 seconds

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST

All lanes : Anti-DAB2 antibody [EPR22881-69] (ab256524) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : LNCaP (human prostate carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 95 kDa why is the actual band size different from the predicted?

Negative control: LNCaP (PMID: 26143155). 

The actual band size higher than predicted is mainly caused by phosphorylation (PMID: 28876503). 

The molecular weight observed is consistent with what has been described in the literature (PMID: 24648493).

Exposure time: 15 seconds

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST

DAB2 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab256524 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256524 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg

Lane 2: ab256524 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256490 in HeLa whole cell lysate.

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

Exposure time: 30 seconds

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling DAB2 with ab256524 at 1/500 dilution (0.14ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in mouse kidney (PMID: 30052485) is observed. The section was incubated with ab256524 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded Human bladder cancer tissue labeling DAB2 with ab256524 at 1/500 dilution (0.14ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human bladder cancer (PMID:17671122) is observed. The section was incubated with ab256524 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling DAB2 with ab256524 at 1/500 dilution (0.14ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human placenta (PMID:27072056) is observed. The section was incubated with ab256524 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control/ Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).