Anti-Cytokeratin 5 antibody [EPR1600Y]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

IHC image of Cytokeratin 5 staining in a section of formalin-fixed paraffin-embedded normal human cervix* performed on a Leica Biosystems BOND® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab78569, 0.1ug/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 haematoxylin 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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling Cytokeratin 5 with purified ab75869 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (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) was used as the unlabeled control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Unpurified ab75869 at 1/100 dilution staining Cytokeratin 5 in human squamous cervical carcinoma by Immunohistochemistry, Paraffin-embedded tissue.

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Fluorescent immunohistochemical analysis of paraffin-embedded human cervical carcinoma tissue using unpurified ab75869. Green-CK5 red-PI.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Immunohistochemical analysis of paraffin embedded human tonsil tissue section labelling Cytokeratin 5 with purified ab75869 at dilution of 1/1000. The secondary antibody used was HRP-conjugated Goat Anti-Rabbit IgG H&L (ab97051) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells labelling Cytokeratin 5 with purified ab75869 at 1/100. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary. Nuclei couterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used, followed by anti-mouse secondary antibody (ab150120). For negative control 2, mouse primary antibody (ab7291) was used followed by anti-rabbit secondary antibody (ab150077).

• WB

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Western blot - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Cytokeratin 5 antibody [EPR1600Y] (ab75869) at 1/50000 dilution

Lane 1:

A431 (human epidermoid carcinoma) whole cell lysate at 20 µg

Lane 2:

HACAT (human keratinocyte) whole cell lysate at 20 µg

Lane 3:

Human skin tissue lysate at 20 µg

Lane 4:

Human fetal tonsil tissue lysate at 20 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 62 kDa

Observed band size: 62 kDa

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• WB

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Western blot - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

All lanes:

Western blot - Anti-Cytokeratin 5 antibody [EPR1600Y] (ab75869) at 1/10000 dilution

All lanes:

A431 cell lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 62 kDa

Observed band size: 62 kDa

false

• WB

Lab

Western blot - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cytokeratin 5 antibody [EPR1600Y] (ab75869) at 1/1000 dilution

Lane 1:

N-GST tagged full-length recombinant human Cytokeratin 6A Protein at 10 ng

Lane 2:

N-GST tagged full-length recombinant human Cytokeratin 5 protein at 10 ng

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

Observed band size: 87 kDa

false

Exposure time: 10s

• OI-RD Scanning

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OI-RD Scanning - Anti-Cytokeratin 5 antibody [EPR1600Y] (AB75869)

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.