Anti-CENPB antibody [EPR24047-64] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling CENPB with ab259855 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 µg/mL dilution (Green). Confocal image showing centromere staining in HeLa cell line. is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5µg/mL dilution (Red). The Nuclear counterstain was DAPI (Blue).

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.

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.

• IP

Supplier Data

Immunoprecipitation - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.

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 : ab259855 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.

All lanes:

Immunoprecipitation - Anti-CENPB antibody [EPR24047-64] (<a href='/en-us/products/primary-antibodies/cenpb-antibody-epr24047-64-ab259855'>ab259855</a>)

Predicted band size: 65 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.

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 : ab259855 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

All lanes:

Immunoprecipitation - Anti-CENPB antibody [EPR24047-64] (<a href='/en-us/products/primary-antibodies/cenpb-antibody-epr24047-64-ab259855'>ab259855</a>)

Predicted band size: 65 kDa

false

• WB

Lab

Western blot - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation.

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

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.

All lanes:

Western blot - Anti-CENPB antibody [EPR24047-64] (<a href='/en-us/products/primary-antibodies/cenpb-antibody-epr24047-64-ab259855'>ab259855</a>) at 1/1000 dilution

Lane 1:

Wild-type A431 (human epidermoid carcinoma epithelial cell) whole cell lysate

Lane 2:

CENPB knockout A431 whole cell lysate

Lane 3:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 65 kDa

false

• WB

Lab

Western blot - Anti-CENPB antibody [EPR24047-64] - BSA and Azide free (AB284394)

This data was developed using ab259855, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 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.

All lanes:

Western blot - Anti-CENPB antibody [EPR24047-64] (<a href='/en-us/products/primary-antibodies/cenpb-antibody-epr24047-64-ab259855'>ab259855</a>) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

HEK-293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 2:

Western blot - Human CENPB knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-cenpb-knockout-a-431-cell-line-ab274919'>ab274919</a>)

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Secondary

Lane 1:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated

Lanes 2 - 4:

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 1/50000 dilution

Lanes 2 - 3:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/50000 dilution

Lane 4:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 65 kDa

false