Anti-BMAL1 antibody [EPR23696-22]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) labeling BMAL1 with ab230822 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) (ab150077) secondary antibody at 1/1000 dilution (green).

Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 was used as a counterstain.

The nuclear counterstain is DAPI (blue).

Confocal image showing positive staining in PC-3 cell line.

Negative Control : Raji (PMID : 29230015).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

Lab

Immunoprecipitation - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

ab230822 at 1/30 (2μg in 0.35mg lysates) immunoprecipitating BMAL1 in PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input) : PC-3 whole cell lysate (10μg)

Lane 2 (+) : ab230822 + PC-3 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab230822 in PC-3 whole cell lysate.

For western blotting, ab230822 at 1/1000 dilution (0.58 μg/mL) and ab131366 VeriBlot for IP (HRP) at 1/5000 was used for detection.

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

Fresh lysates were used in this IP.
The molecular weight observed is consistent with what has been described in the literature (PMID : 28332504).

All lanes:

Immunoprecipitation - Anti-BMAL1 antibody [EPR23696-22] (ab230822)

Predicted band size: 68 kDa

Observed band size: 75 kDa

false

Exposure time: 50s

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

Immunohistochemical analysis of paraffin-embedded Mouse hippocampus tissue labeling BMAL1 with ab230822 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in mouse hippocampus (PMID : 20382135). The section was incubated with ab230822 for 20 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Antigen retrieval was heat mediated with Citrate buffer (pH 6.0, epitope retrieval solution 1) 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-BMAL1 antibody [EPR23696-22] (AB230822)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling BMAL1 with ab230822 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in mouse cardiac muscle (PMID : 10760301). The section was incubated with ab230822 for 20 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Antigen retrieval was heat mediated with Citrate buffer (pH 6.0, epitope retrieval solution 1) 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-BMAL1 antibody [EPR23696-22] (AB230822)

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling BMAL1 with ab230822 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in rat pancreas (PMID : 29396463). The section was incubated with ab230822 for 20 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Antigen retrieval was heat mediated with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

• IP

Lab

Immunoprecipitation - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

ab230822 at 1/30 (2μg in 0.35mg lysates) immunoprecipitating BMAL1 in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate.

Lane 1 (input) : NIH/3T3 whole cell lysate (10μg)

Lane 2 (+) : ab230822 + NIH/3T3 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab230822 in NIH/3T3 whole cell lysate.

For western blotting, ab230822 at 1/1000 dilution (0.58 μg/mL) and ab131366 VeriBlot for IP (HRP) at 1/5000 was used for detection.

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

Fresh lysates were used in this IP.

All lanes:

Immunoprecipitation - Anti-BMAL1 antibody [EPR23696-22] (ab230822)

Predicted band size: 68 kDa

Observed band size: 75 kDa

false

Exposure time: 50s

• WB

Lab

Western blot - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

Western blot : Rabbit monoclonal [EPR23696-22] to BMAL1 ab230822 staining at 1/500 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta.

A band was observed at 69-75 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in ARNTL knockout MCF7 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.

Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-BMAL1 antibody [EPR23696-22] (ab230822) at 1/500 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

ARNTL knockout MCF7 at 20 µg

Lane 2:

Western blot - Human ARNTL knockout MCF7 cell line (<a href='/ko/products/cell-lines/human-arntl-knockout-mcf7-cell-line-ab289293'>ab289293</a>) at 20 µg

Lane 3:

Wild-type HeLa at 20 µg

Lane 4:

ARNTL knockout HeLa <a href='/ko/products/cell-lines/human-arntl-bmal1-knockout-hela-cell-line-ab264701'>ab264701</a> at 20 µg

Lane 5:

Raji at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 69-75 kDa

Observed band size: 69-75 kDa

false

• WB

Lab

Western blot - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The molecular weight observed is consistent with what has been described in the literature (PMID : 28332504). Exposure time : 70 seconds

All lanes:

Western blot - Anti-BMAL1 antibody [EPR23696-22] (ab230822) at 1/1000 dilution

All lanes:

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

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

Western blot : Rabbit monoclonal [EPR23696-22] to BMAL1 ab230822 staining at 1/500 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 69 kDa in Wild-type A549 and HeLa cell lysates with no signal observed at this size in ARNTL knockout A549 cell line and ARNTL knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-BMAL1 antibody [EPR23696-22] (ab230822) at 1/500 dilution

Lane 1:

Wild-type A549 cell lysates at 20 µg

Lane 2:

Western blot - Human ARNTL knockout A549 cell line (ab288835) at 20 µg

Lane 3:

Wild-type HeLa ab255928 cell lysates at 20 µg

Lane 4:

Western blot - Human ARNTL (BMAL1) knockout HeLa cell line (<a href='/ko/products/cell-lines/human-arntl-bmal1-knockout-hela-cell-line-ab264701'>ab264701</a>) at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 69 kDa

Observed band size: 69 kDa

false

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HepG2 (human hepatocellular carcinoma epithelial cell) cells and 5 µg of ab230822 [EPR23696-22]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HepG2 (human hepatocellular carcinoma epithelial cell) cells and 5 µg of ab230822 [EPR23696-22]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 HepG2 (human hepatocellular carcinoma epithelial cell) cells and 5 µg of ab230822 [EPR23696-22]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-BMAL1 antibody [EPR23696-22] (AB230822)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The molecular weight observed is consistent with what has been described in the literature (PMID : 28332504). Exposure time : 70 seconds

All lanes:

Western blot - Anti-BMAL1 antibody [EPR23696-22] (ab230822) at 1/1000 dilution

Lane 1:

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

Lane 2:

Rat liver tissue lysate 20 at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 68 kDa

Observed band size: 75 kDa

false