Recombinant Anti-UFC1 antibody [EPR15014-102] (ab189252)

Lanes 1- 2: Merged signal (red and green). Green - ab189252 observed at 20 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab189252 was shown to react with UFC1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266814 (knockout cell lysate ab257781) was used. Wild-type HEK-293T and UFC1 HEK-293T KO 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. ab189252 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 formalin-fixed, paraffin-embedded Human colon tissue labeling UFC1 with ab189252 at 1/100 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Lanes 1 - 4: Merged signal (red and green). Green - ab189252 observed at 20 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab189252 was shown to react with UFC1 in wild-type HAP1 cells in western blot. Loss of signal was observed when UFC1 knockout sample was used. Wild-type HAP1 and UFC1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab189252 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human transitional cell carcinoma of bladder tissue labeling UFC1 with ab189252 at 1/100 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Western blot analysis of immunoprecipitation pellet from MCF7cell lysate immunoprecipitated using ab189252 at 1/50 dilution (lane 1) or PBS control (lane 2).
Secondary: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1500 dilution.