All lanes : Anti-Cytokeratin 8 antibody [EP1628Y] - Cytoskeleton Marker (ab53280) at 1/10000 dilution

Lane 1 : A431 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : KRT8 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 54 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab53280 observed at 55 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab53280 was shown to react with Cytokeratin 8 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255400 (knockout cell lysate ab263785) was used. Wild-type and Cytokeratin 8 knockout samples were subjected to SDS-PAGE. ab53280 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 (For unpurified use at 1/25,000 - 1/50,000) dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunocytochemistry/ Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling Cytokeratin 9 with Purified ab53280 at 1:500 dilution. Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Alexa Fluor^® 488 (ab192467) and Alexa Fluor^® 647 (ab192468) conjugated versions are available for this clone.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue sections labeling Cytokeratin 8 with Purified ab53280 at 1:250 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cytokeratin 8 with purified ab53280 at 1:20 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Alexa Fluor^® 488 (ab192467) and Alexa Fluor^® 647 (ab192468) conjugated versions are available for this clone.

All lanes : Anti-Cytokeratin 8 antibody [EP1628Y] - Cytoskeleton Marker (ab53280) at 1/10000 dilution (purified)

Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
Lane 2 : Human breast cancer lysates
Lane 3 : HaCaT (Human skin keratinocyte) whole cell lysates
Lane 4 : Mouse colon lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 54 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

ab53280 (purified) at 1:20 dilution (0.2μg) immunoprecipitating Cytokeratin 8 in HeLa whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate, 10μg
Lane 2 (+): ab53280 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab53280 in HeLa whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Panx1^-/- mice have normal mammary gland epithelial differentiation at lactation

Immunofluorescent analysis of luminal epithelial marker keratin 8 (green) and myoepithelial marker keratin14 (red) revealed a similar staining pattern in Panx1^-/- mice compared to control mice during lactation. Paraffin-embedded tissue samples.

Hoescht (blue) denotes nuclei. N = 6. Scale bars = 50 um. 

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling Cytokeratin 8 with purified ab53280 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised by 0.1% tritonX-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Unpurified ab53280 (1:250) staining human Cyotkeratin 8 in human breast adenocarcinoma tissue by immunohistochemistry using paraffin embedded tissue.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling Cytokeratin 8 with Purified ab53280 at 1:250 dilution. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Anti-Cytokeratin 8 antibody [EP1628Y] - Cytoskeleton Marker (ab53280) at 1/50000 dilution (unpurified) + A431 cell lysate at 10 µg

Secondary
Goat anti-Rabbit HRP labeled at 1/2000 dilution

Predicted band size: 54 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

Unpurified ab53280 staining Cytokeratin 8 in Mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formalin and blocked with 10% serum for 20 minutes at 23°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/75 in TBS + 1% BSA) for 1 hour at 23°C. A Biotin-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

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Immunofluorescent staining of HeLa cells using unpurified ab53280 (1:100).

Image: Courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical School

Sample: mouse embryonic stem cell-differentiated embryoid bodies (EBs)

Preparation:

Fix in 3%PFA in PBS for 30 min at RTIncubate in 7.5% sucrose-PBS for 3h at RTIncubate in 15% sucrose-PBS at 4 degree Celsius overnightEmbed the EBs in tissue-Tek OCT compoundCut frozen sections to 4-20 µm thickness

Primary antibody 1: Rabbit anti cytokeratin 8 (unpurified ab53280), 1:100

Primary antibody 2: Rat anti-perlecan, 1:100
Secondary antibody 1: Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150081), 1:200

Secondary antibody 2: Goat polyclonal Secondary Antibody to Rat IgG - H&L (Cy5®) pre-adsorbed (ab150081), 1:200
Nuclei were counterstained with DAPI

 

Fluorescent immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue using unpurified ab53280. Green-CK8 red-PI.

Fluorescent immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using unpurified ab53280. Green-CK8 red-PI

Fluorescent immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue using unpurified ab53280. Green-CK8 red-PI.

Fluorescent immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue using unpurified ab53280. Green-CK8 red-PI.

Overlay histogram showing HeLa cells stained with unpurified ab53280 (red line). The cells were fixed with 2% PFA (room temperature, 30 min) and then permeabilized with 1% FACS permeabilizing solution for 30 min. The cells were then incubated in 3% FBS in 1X PBS followed by the antibody (ab53280, 1/20 dilution) for 1 hour at room temperature. The cells were then incubated for 30 min at room temperature with the secondary antibody. An isotype control antibody (black line) was used and an unlabelled sample (blue line) was also used as a control.

Equilibrium disassociation constant (K_D)
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