Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Intracellular 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Intracellular 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labeling RBBP7 with ab259957 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 3 μg of ab259957 [EPR23796-74]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. At time of publication of this image, ChIP-seq was not widely characterised in HeLa for this antibody. For any questions, please contact Abcam Technical Support. Additional screenshots of mapped reads can be downloaded here.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 8 μg of ab259957 [EPR23796-74]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here.

• IP

Unknown

Immunoprecipitation - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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 : ab259957 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.

All lanes:

Immunoprecipitation - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (ab259957)

Predicted band size: 48 kDa

Observed band size: 48 kDa

false

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Intracellular 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling RBBP7 with ab259957 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling RBBP7 with ab259957 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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.

• IP

Unknown

Immunoprecipitation - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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 : ab259957 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.

All lanes:

Immunoprecipitation - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (ab259957)

Predicted band size: 48 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

Lanes 1 - 2 : Merged signal (red and green). Green - ab259957 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab259957 was shown to react with RBBP7 in wild-type HeLa cells in Western blot with loss of signal observed in RBBP7 knockout cell line ab264677 (RBBP7 knockout cell lysate ab258628). Wild-type HeLa and RBBP7 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab259957 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (ab259957) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RBBP7 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RBBP7 (RbAp46) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rbbp7-rbap46-knockout-hela-cell-line-ab264677'>ab264677</a>)

Predicted band size: 48 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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.

All lanes:

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (ab259957) at 1/2000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

RBBP7 (RbAp46) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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: 48 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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

Exposure times : 8 seconds.

All lanes:

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (ab259957) at 1/1000 dilution

Lane 1:

Jurkat (human t cell leukemia t lymphocyte) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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: 48 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (AB259957)

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.

All lanes:

Western blot - Anti-RBBP7 antibody [EPR23796-74] - ChIP Grade (ab259957) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse spleen tissue lysate at 20 µg

Lane 5:

Rat heart tissue lysate at 20 µg

Lane 6:

Rat kidney tissue lysate at 20 µg

Lane 7:

Rat spleen tissue lysate at 20 µg

Lane 8:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 9:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 10:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 11:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

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: 48 kDa

Observed band size: 48 kDa

false