Anti-Cytokeratin 13 antibody [AE8] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 13 antibody [AE8] - BSA and Azide free (AB269574)

IHC image of Cytokeratin 13 staining in a section of formalin-fixed paraffin-embedded normal human tonsil* performed on a Leica BOND^TM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16112, 0.05 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This image was produced using the same antibody clone but in a different formulation ab16112, PBS and sodium azide.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Cytokeratin 13 antibody [AE8] - BSA and Azide free (AB269574)

IHC image of Cytokeratin 13 staining in a section of frozen normal human tonsil*.

The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab16112 at 1μg/ml. The section was then incubated with ab150117 (Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488), 1/1000)) (shown in green) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM. The secondary-only control insert image is taken from an identical assay without primary antibody. The section was then mounted using Fluoromount®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

This image was produced using the same antibody clone but in a different formulation ab16112, PBS and sodium azide.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 13 antibody [AE8] - BSA and Azide free (AB269574)

ab16112 staining Cytokeratin 13 in A431 cells. The cells were fixed with Methanol (5min), permeabilized with 0.1%PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab16112 at 5μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This image was produced using the same antibody clone but in a different formulation ab16112, PBS and sodium azide.

• WB

Supplier Data

Western blot - Anti-Cytokeratin 13 antibody [AE8] - BSA and Azide free (AB269574)

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 3% milk before ab16112 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 1ug/ml concentration and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

This image was produced using the same antibody clone but in a different formulation ab16112, PBS and sodium azide.

All lanes:

Western blot - Anti-Cytokeratin 13 antibody [AE8] (<a href='/en-us/products/primary-antibodies/cytokeratin-13-antibody-ae8-ab16112'>ab16112</a>) at 1 µg/mL

All lanes:

A431 whole cell lysate at 20 µg

Predicted band size: 50 kDa

false

• WB

Lab

Western blot - Anti-Cytokeratin 13 antibody [AE8] - BSA and Azide free (AB269574)

This image was produced using the same antibody clone but in a different formulation ab16112, PBS and sodium azide.

False colour image of Western blot : Anti-Cytokeratin 13 antibody [AE8] staining at 1 μg/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab16112 was shown to bind specifically to Cytokeratin 13. A band was observed at 51 kDa in wild-type A431 cell lysates with no signal observed at this size in Krt13 knockout cell line ab269483 (knockout cell lysate ab269647). To generate this image, wild-type and Krt13 knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-Cytokeratin 13 antibody [AE8] (<a href='/en-us/products/primary-antibodies/cytokeratin-13-antibody-ae8-ab16112'>ab16112</a>) at 1 µg/mL

Lane 1:

Wild-type A431 cell lysate at 20 µg

Lane 2:

KRT13 knockout A431 cell lysate at 20 µg

Lane 2:

Western blot - Human KRT13 knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-krt13-knockout-a-431-cell-line-ab269483'>ab269483</a>)

Predicted band size: 50 kDa

Observed band size: 51 kDa

false