Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling NRF1 with purified ab175932 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labeling NRF1 with unpurified ab175932 at a dilution of 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling NRF1 with unpurified ab175932 at a dilution of 1/50.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Intracellular Flow Cytometry analysis of 293T cells labelling NRF1 with purified ab175932 at a dilution of 1/150 (red). Cells were fixed with 80% methanol. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue labeling NRF1 with unpurified ab175932 at a dilution of 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Intracellular flow cytometric analysis of permeabilized 293T cells labeling NRF1 with unpurified ab175932 at a dilution of 1/10 (red) compared to a negative control (rabbit IgG,green).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cevical carcinoma tissue labelling NRF1 with purified ab175932 at a dilution of 1/100. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IP

Supplier Data

Immunoprecipitation - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

ab175932 (unpurified) at a dilution of 1/10 immunoprecipitating NRF1 in 293T cell lysate.

All lanes:

Immunoprecipitation - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932)

Predicted band size: 54 kDa

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• IP

Lab

Immunoprecipitation - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

ab175932 (purified) at a dilution of 1/50 immunoprecipitating NRF1 in 293T whole cell lysate.

Lane 1 (input) : 293T whole cell lysate (10μg)

Lane 2 (+) : ab175932 + 293T whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab175932 in 293T whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932)

Predicted band size: 54 kDa

Observed band size: 68 kDa

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• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

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 ab175932 [EPR5554(N)]. 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.

• ChIP

Unknown

ChIP - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Chromatin was prepared from Hela cells according to the Abcam Dual X-ChIP protocol. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab175932 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• ChIP

Unknown

ChIP - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Chromatin was prepared from NIH/3T3 treated with MG-132(2uM 16h) cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab175932 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• WB

Lab

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/10000 dilution

All lanes:

HEK293 whole cell lysate at 20 µg

Secondary

All lanes:

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 1/100000 dilution

Predicted band size: 54 kDa

Observed band size: 68 kDa

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• WB

Lab

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/10000 dilution

All lanes:

HeLa whole cell lysate at 20 µg

Secondary

All lanes:

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 1/100000 dilution

Predicted band size: 54 kDa

Observed band size: 68 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5 µg of ab175932 [EPR5554(N)]. 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 ChIP data was conducted on chromatin 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 ab175932. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 2.5x10^5 HeLa cells and 5μg of ab175932 [EPR5554(N)]. The resulting DNA was sequenced on the NovaSeq 6000 (PE150). Additional screenshots of mapped reads can be downloaded here.

• WB

Lab

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/5000 dilution

Lane 1:

Mouse heart tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat heart tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

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 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 68 kDa

false

• WB

Supplier Data

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (AB175932)

All lanes:

Western blot - Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/1000 dilution

Lane 1:

MCF-7 cell lysate at 10 µg

Lane 2:

Hela cell lysate at 10 µg

Lane 3:

Human fetal heart tissue lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Predicted band size: 54 kDa

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