Recombinant Anti-CENPB antibody [EPR24047-64] (ab259855)

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 µg/mL dilution.

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

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Fresh lysates are preferred in this product.

Exposure time: 3 minutes 

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-4: Merged signal (red and green). Green - ab259855 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

ab259855 Anti-CENPB antibody [EPR24047-64] was shown to specifically react with CENPB in wild-type A431 cells. Loss of signal was observed when the knockout cell line ab274919 (knockout cell lysate ab274977) was used. Wild-type and CENPB knockout samples were subjected to SDS-PAGE. ab259855 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight 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 10000 dilution for 1 hour at room temperature before imaging.

Fresh lysates are preferred in this product. 

CENPB was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg with ab259855 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259855 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug
Lane 2: abab259855 IP in NIH/3T3 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259855 in NIH/3T3 whole cell lysate

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

Exposure time: 3 minutes

CENPB was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg with ab259855 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259855 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug
Lane 2: abab259855 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259855 in HeLa whole cell lysate

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

Exposure time: 15 seconds

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling CENPB with ab259855 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma tissue labelling CENPB with ab259855 at 1/100 (5.39 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on centromere in human ovarian carcinoma (PMID: 12839935).The section was incubated with ab259855 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labelling CENPB with ab259855 at 1/100 (5.39 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on centromere in human gastric cancer (PMID: 12839935). The section was incubated with ab259855 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labelling CENPB with ab259855 at 1/100 (5.39 μg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on centromere in human pancreas (PMID: 12839935). The section was incubated with ab259855 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.