重组Anti-NRF1抗体[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 downloaded here.

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

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.

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).

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. 

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.

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

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

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). 

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.

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.