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).

• 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).

• 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='/en-us/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 1/20,000 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='/en-us/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='/en-us/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

• 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='/en-us/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

• 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

• 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