Anti-Cytochrome P450 Reductase 抗体 [EPR14479(B)] (ab180597)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labelling Cytochrome P450 Reductase with ab180597 at 1/1000 (0.1 μg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).The section was counterstained with Hematoxylin. Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Postive staining on mouse kidney. The section was incubated with ab180597 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: Lane 1-3: 26 seconds; Lane 4-7: 75 seconds.

The identities of the lower MW bands between 37 and 60kDa are unknown.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: Lane 1-3: 26 seconds; Lane 4-7: 75 seconds.

The identities of the lower MW bands between 37 and 60kDa are unknown.

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.

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.

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.

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.

Intracellular Flow Cytometry analysis of HeLa cells using ab180597 at a 1/10 dilution (red) and a rabbit IgG as negative control (green). Goat anti rabbit IgG (FITC) secondary usedat a 1/150 dilution.