All lanes : Anti-RBBP7 antibody [EPR23796-74] (ab259957) at 1/2000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : RBB7 (RbAp46) knockout HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 48 kDa
Observed band size: 48 kDa

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

ab259957 was shown to specifically react with RBBP7 in wild-type HAP1 cells as signal was lost in RBB7 (RbAp46) knockout cells.

Wild-type and RBB7 (RbAp46) knockout samples were subjected to SDS-PAGE. ab259957 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/2000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/50,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc^™ MP instrument using the ECL technique.

Exposure time: 10 seconds. 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RBBP7 (RbAp46) KO HAP1 cells labelling RBBP7 with ab259957 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing no staining in RBBP7 (RbAp46) KO HAP1 cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

All lanes : Anti-RBBP7 antibody [EPR23796-74] (ab259957) at 1/1000 dilution

Lane 1 : Jurkat (human t cell leukemia t lymphocyte) whole cell lysate
Lane 2 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate
Lane 4 : LNCaP (human prostate carcinoma epithelial cell) whole cell lysate
Lane 5 : F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 48 kDa
Observed band size: 48 kDa

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

Exposure times: 8 seconds. 

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling RBBP7 with abab259957 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in mouse lung (PMID: 19655816). The section was incubated with ab259957 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling RBBP7 with ab259957 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing mainly nuclear staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized parental HAP1 (Wildtype control Human chronic myelogenous leukemia near-haploid cell line (Right) / RBBP7 (RbAp46) KO HAP1 (Left) cells labelling RBBP7 with ab259957 at 1/600 dilution (0.1ug) (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

RBBP7 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate  with ab259957 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259957 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug

Lane 2: abab259957 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259957 in NIH/3T3 whole cell lysate

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

Exposure time: 24 seconds.

 

RBBP7 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab259957 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259957 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 10 ug

Lane 2: abab259957 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259957 in HeLa whole cell lysate

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

Exposure time: 24 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling RBBP7 with ab259957 at 1/600 dilution (0.1ug) (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labeling RBBP7 with abab259957 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in human lung cancer (PMID: 19655816).The section was incubated with ab259957 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Hela (Human cervix adenocarcinoma epithelial cell) cells labelling RBBP7 with ab259957 at 1/600 dilution (0.1ug) (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling RBBP7 with ab259957 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing mainly nuclear staining in HeLa cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling RBBP7 with abab259957 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in rat lung (PMID: 19655816). The section was incubated with ab259957 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-RBBP7 antibody [EPR23796-74] (ab259957) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Mouse liver tissue lysate
Lane 4 : Mouse spleen tissue lysate
Lane 5 : Rat heart tissue lysate
Lane 6 : Rat kidney tissue lysate
Lane 7 : Rat spleen tissue lysate
Lane 8 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 9 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 10 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 11 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 48 kDa
Observed band size: 48 kDa

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

Exposure times: Lanes 1-3: 3 minutes; Lane 4: 3 seconds; Lanes 5-6: 3 minutes; Lane 7-11: 3 seconds.