All lanes : Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/5000 dilution (purified)

Lane 1 : Mouse heart tissue lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Rat heart tissue lysate
Lane 4 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 54 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?



Blocking and dilution buffer: 5% NFDM/TBST

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).

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.

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

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.

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

Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/10000 dilution (purified) + HEK293 whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?



Blocking and dilution buffer: 5% NFDM/TBST

Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/10000 dilution (purified) + HeLa whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?



Blocking and dilution buffer: 5% NFDM/TBST

All lanes : Anti-NRF1 antibody [EPR5554(N)] - ChIP Grade (ab175932) at 1/1000 dilution (unpurified)

Lane 1 : MCF-7 cell lysate
Lane 2 : Hela cell lysate
Lane 3 : Human fetal heart tissue lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 54 kDa

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.

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.