All lanes : Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] (ab180597) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : POR knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab180597 observed at 80 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab180597 was shown to react with Cytochrome P450 Reductase in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266889 (knockout cell lysate ab257596) was used. Wild-type HCT116 and POR knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab180597 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin embedded Human infiltrating duct carcinoma of breast tissue labeling Cytochrome P450 Reductase with ab180597 at a 1/150 dilution. HRP Polymer for Rabbit IgG secondary used. Counterstained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

All lanes : Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] (ab180597) at 1/10000 dilution

Lane 1 : Wild-type HeLa lysate
Lane 2 : Cytochrome P450 Reductase knockout HeLa lysate
Lane 3 : HepG2 lysate
Lane 4 : A431 lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Lanes 1-4: Merged signal (red and green). Green - ab180597 observed at 75 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab180597 Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] was shown to specifically react with Cytochrome P450 Reductase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264996 (knockout cell lysate ab257595) was used. Wild-type and Cytochrome P450 Reductase knockout samples were subjected to SDS-PAGE. ab180597 and Anti-GAPDH antibody [6C5] - Loading Control?(ab8245) were incubated overnight at 4^°C at 1 in 10000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

 

All lanes : Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] (ab180597) at 1/10000 dilution

Lane 1 : HeLa lysate
Lane 2 : MCF7 lysate
Lane 3 : A431 lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated at 1/1000 dilution

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling Cytochrome P450 Reductase with purified ab180597 at 1/500 dilution. Cells were fixed with 100% methanol. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] (ab180597) at 1/50000 dilution + HepG2 lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated at 1/1000 dilution

Immunofluorescence analysis of Hela cells (fixative 4% paraformaldehyde) labeling Cytochrome P450 Reductase with ab180597 at a 1/100 dilution (red), and counterstained with Dapi (blue). Goat anti rabbit IgG (Dylight 555) secondary used at a 1/200 diution.

Immunofluorescence analysis of MCF7 cells (fixative 4% paraformaldehyde) labeling Cytochrome P450 Reductase with ab180597 at a 1/100 dilution (green), and counterstained with Dapi (blue). Goat anti rabbit IgG (Dylight 555) secondary used at a 1/200 diution.

Flow cytometry analysis of HeLa cells using ab1805937 at a 1/10 dilution (red) and a rabbit IgG as negative control (green). Goat anti rabbit IgG (FITC) secondary used at a 1/150 dilution.