Recombinant Anti-SDHB antibody [EPR10880] (ab175225)

Lanes 1 - 3: Merged signal (red and green). Green - ab175225 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab175225 was shown to recognize SDHB in wild-type HEK 293 cells as signal was lost at the expected MW in SDHB knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SDHB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab175225 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/50000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling SDHB with purified ab175225 at a dilution of 1/2000. Heat mediated antigen retrieval was performed using Tris/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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labeling SDHB with unpurified ab175225 at a dilution of 1/100.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human mesenchymoma tissue labelling SDHB with purified ab175225 at a dilution of 1/2000. Heat mediated antigen retrieval was performed using Tris/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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labelling SDHB with purified ab175225 at a dilution of 1/2000. Heat mediated antigen retrieval was performed using Tris/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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling SDHB with purified ab175225 at a dilution of 1/2000. Heat mediated antigen retrieval was performed using Tris/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.

Intracellular Flow Cytometry analysis ofA431 cells labelling SDHB with purified ab175225 at a dilution of 1/200 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^®488-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. 

ab175225 (purified) at a dilution of 1/60 immunoprecipitating SDHB in Jurkat whole cell lysate.

Lane 1 (input): Jurkat whole cell lysate (10µg)

Lane 2 (+): ab175225 + Jurkat whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab175225 in Jurkat 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labeling SDHB with unpurified ab175225 at a dilution of 1/100.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human carotid paraganglioma tissue labeling SDHB with unpurified ab175225 at a dilution of 1/100.

Intracellular flow cytometric analysis of permeabilized Jurkat cellslabeling SDHB with unpurified ab175225 at a dilution of 1/10 (red) compared toa rabbit IgG negative control (green).