Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

ab76126 showing negative staining in human breast carcinoma 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 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

Fluorescent immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue using ab76126. Green-CK20 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 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

ab76126 showing positive staining in human colonic adenocarcinoma tissue.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

ab76126 showing negative staining in human hepatocellular carcinoma tissue.

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

ab76126 staining Cytokeratin 20 in HT-29 cells, with negative expression in MCF7 cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% 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 ab76126 at 0.1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

This product also work with 4% formaldehyde (10 min) fixation under the same testing conditions.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

Overlay histogram showing LoVo (Human colorectal adenocarcinoma cell line) cells stained with ab76126 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab76126, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

ab76126 showing positive staining in human urinary bladder transitional carcinoma tissue.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

Immunocytochemistry/Immunofluorescence analysis of HT-29 (human colorectal adenocarcinoma) cells labelling Cytokeratin 20 with purified ab76126 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control : PBS was used instead of the primary antibody as the negative control.

• WB

Lab

Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of human small intestine lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab76126 (1/10,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG with minimal cross-reactivity with human IgG at 1/2000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab76126 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant for temperatures ranging between 4°C, 22°C and 37°C, demonstrating that antibody activity is not affected under these conditions.

All lanes:

Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (ab76126) at 1/10000 dilution

All lanes:

Human small intestine lysate at 15 µg with NDFM/TBST

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 1/2000 dilution

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Exposure time: 10s

• WB

Lab

Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

False colour image of Western blot : Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab76126 was shown to bind specifically to Cytokeratin 20. 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-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (ab76126) at 1/10000 dilution

Lane 1:

HT-29 cell lysate at 20 µg

Lane 2:

DLD-1 cell lysate at 20 µg

Lane 3:

SW480 cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Lane 5:

MCF7 cell lysate at 20 µg

Predicted band size: 48 kDa

Observed band size: 45 kDa,45-50 kDa

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• WB

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Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

Primary : ab76126, 1/50000 dilution
Sample : human small intestine lysate, 10 ug
Secondary : goat anti-rabbit-HRP, 1/1000 dilution

All lanes:

Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (ab76126) at 1/50000 dilution

All lanes:

human small intestine lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit-HRP at 1/1000 dilution

Predicted band size: 48 kDa

Observed band size: 44 kDa

false

• WB

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Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

Primary : ab76126, 1/50000 dilution
Sample : ab73640, 0.01 ug
Secondary : ab97080, 1/5000 dilution

All lanes:

Western blot - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (ab76126) at 1/50000 dilution

All lanes:

Recombinant Human Cytokeratin 20 protein (<a href='/en-us/products/unavailable/recombinant-human-cytokeratin-20-protein-ab73640'>ab73640</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 48 kDa

true

Exposure time: 20s

• OI-RD Scanning

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OI-RD Scanning - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (AB76126)

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.