Recombinant Anti-ARPC5/p16 ARC antibody [EP1551Y] (ab51243)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: p16 ARC knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab51243 observed at 16 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab51243 was shown to specifically react with p16 ARC when p16 ARC knockout samples were used. Wild-type and p16 ARC knockout samples were subjected to SDS-PAGE. Ab51243 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling p16 ARC with purified ab51243 at a dilution of 1/50. 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.

Immunocytochemistry/Immunofluorescence analysis of Neuro-2a cells labelling p16 ARC with purified ab51243 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

ab51243 (purified) at a dilution of 1/20 immunoprecipitating p16 ARC in human fetal brain tissue lysate.

Lane 1 (input): Human fetal brain tissue lysate (10µg)

Lane 2 (+): ab51243 + human fetal brain tissue lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab51243 in human fetal brain tissue lysate.

For western blotting, a HRP-conjugated anti-rabbit specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human uterus adenocarcinoma tissue labelling p16 ARC with unpurified ab51243 at a dilution of 1/50.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.