Anti-DAB2 antibody [EPR22881-69]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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).

• IP

Unknown

Immunoprecipitation - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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

All lanes:

Immunoprecipitation - Anti-DAB2 antibody [EPR22881-69] (ab256524)

Predicted band size: 82 kDa

Observed band size: 95 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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).

• WB

Lab

Western blot - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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:

Western blot - Anti-DAB2 antibody [EPR22881-69] (ab256524) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

Caco-2 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 82 kDa

Observed band size: 95 kDa

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• WB

Lab

Western blot - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

DAB2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human DAB2 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-dab2-knockout-hela-cell-line-ab265731'>ab265731</a>)

Lane 3:

Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 82 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-DAB2 antibody [EPR22881-69] (AB256524)

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

All lanes:

Western blot - Anti-DAB2 antibody [EPR22881-69] (ab256524) at 1/1000 dilution

Lane 1:

PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

LNCaP (human prostate carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 82 kDa

Observed band size: 95 kDa

false