Recombinant Anti-ELK1 antibody [E277] (ab32106)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: ELK1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa positive whole cell lysate (21 µg)
Lane 4: MCF7 negative whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32106 observed at 55 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab32106 was shown to specifically react with ELK1 in wild-type HAP1 cells as signal was lost in ELK1 knockout cells. Wild-type and ELK1 knockout samples were subjected to SDS-PAGE. Ab32106 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of A549 (human lung carcinoma epithelial cell) cells labeling ELK1 with purified ab32106 at 1/500 dilution (3.8 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain.

Immunohistochemical analysis of paraffin embedded human breast carcinoma using ab32106 at a dilution of 1/50

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ELK1 with unpurified ab32106 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Lanes 1- 2: Merged signal (red and green). Green - ab32106 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32106 was shown to react with ELK1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261764 (knockout cell lysate ab256904) was used. Wild-type HeLa and ELK1 knockout HeLa 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. ab32106 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 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.