Anti-Cytokeratin 15 antibody [EPR1614Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

ab52816 at 1/50 dilution staining Cytokeratin 15 in HeLa cells by Immunocytochemistry.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

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.

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-Cytokeratin 15 antibody [EPR1614Y] (ab52816) at 1/10000 dilution

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

KRT15 knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human KRT15 knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-krt15-knockout-a-431-cell-line-ab262484'>ab262484</a>)

Predicted band size: 49 kDa

Observed band size: 50 kDa

false

• WB

Unknown

Western blot - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

All lanes:

Western blot - Anti-Cytokeratin 15 antibody [EPR1614Y] (ab52816) at 1/10000 dilution

All lanes:

fetal thymus lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 49 kDa

Observed band size: 45 kDa

false

• WB

Lab

Western blot - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

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

Exposure time : 5.5 seconds

All lanes:

Western blot - Anti-Cytokeratin 15 antibody [EPR1614Y] (ab52816) at 1/100000 dilution

All lanes:

Mouse skin tissue lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 49 kDa

Observed band size: 45 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Cytokeratin 15 antibody [EPR1614Y] (AB52816)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.