Recombinant Anti-Cytokeratin 15 antibody [EPR1614Y] (ab52816)

Lanes 1 - 2: Merged signal (red and green). Green - ab52816 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab52816 was shown to react with Cytokeratin 15 in wild-type A431 cells in Western blot with loss of signal observed in KRT15 knockout cell line ab263922 (knockout cell lysate ab262484). Wild-type A431 and KRT15 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab52816 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 h at room temperature before imaging.

ab52816 at 1/100 dilution staining Cytokeratin 15 in human squamous cervical carcinoma by Immunohistochemistry, Paraffin embedded tissue.

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

Blocking buffer and concentration: 5% NFDM/TBST
Diluting buffer and concentration: 5% NFDM /TBST

Exposure time: 5.5 seconds

Overlay histogram showing A431 cells stained with ab52816 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52816, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in A431 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Triton used under the same conditions.

Fluorescent immunohistochemical analysis of paraffin-embedded human normal skin tissue using ab52816. Green-CK15 red-PI

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

Fluorescent immunohistochemical analysis of paraffin-embedded human cervical carcinoma tissue using ab52816. Green-CK15 red-PI.

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