Recombinant Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade – BSA and Azide free (ab221792)

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.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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, ab131366 VeriBlot for IP (HRP) was used for detection at 1/1000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175932).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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). 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175932). 

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175932).

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

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175932).

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