Recombinant
RabMAb

Recombinant Anti-Cytokeratin 8 antibody [EP1628Y] - Cytoskeleton Marker (ab53280)

Rabbit recombinant monoclonal Cytokeratin 8 antibody [EP1628Y]. Validated in WB, IP, IHC, Flow Cyt, ICC/IF and tested in Mouse, Human. Cited in 39 publication(s). Independently reviewed in 5 review(s).

Overview

  • Product name

    Anti-Cytokeratin 8 antibody [EP1628Y] - Cytoskeleton Marker
    See all Cytokeratin 8 primary antibodies
  • Description

    Rabbit monoclonal [EP1628Y] to Cytokeratin 8 - Cytoskeleton Marker
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IP, Flow Cyt, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human Cytokeratin 8 aa 300-400 (C terminal). The exact sequence is proprietary.
    Database link: P05787

  • Positive control

    • IHC-P: Human breast adenocarcinona, ovarian carcinoma, breast carcinoima, colon adenocarcinoma, endometrial carcinoma and thyroid carcinoma tissue; mouse liver tissue; ICC/IF: HT-29 and HeLa cells; WB: A431 and HaCaT cell lysates; Human breast cancer lysates and Mouse colon lysate; Flow cyt: HeLa cells.
  • General notes

    A trial size is available to purchase for this antibody.

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab53280 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/10000. Detects a band of approximately 52 kDa (predicted molecular weight: 54 kDa).

For unpurified use at 1/25,000 - 1/50,000.

IP 1/20.

For unpurified use at 1:70.

Flow Cyt 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-P 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/100 - 1/500.

Target

  • Function

    Together with KRT19, helps to link the contractile apparatus to dystrophin at the costameres of striated muscle.
  • Tissue specificity

    Observed in muscle fibers accumulating in the costameres of myoplasm at the sarcolemma membrane in structures that contain dystrophin and spectrin. Expressed in gingival mucosa and hard palate of the oral cavity.
  • Involvement in disease

    Cirrhosis
  • Sequence similarities

    Belongs to the intermediate filament family.
  • Post-translational
    modifications

    Phosphorylation on serine residues is enhanced during EGF stimulation and mitosis. Ser-74 phosphorylation plays an important role in keratin filament reorganization.
    O-glycosylated. O-GlcNAcylation at multiple sites increases solubility, and decreases stability by inducing proteasomal degradation.
    O-glycosylated (O-GlcNAcylated), in a cell cycle-dependent manner.
  • Cellular localization

    Cytoplasm. Nucleus, nucleoplasm. Nucleus matrix.
  • Information by UniProt
  • Database links

  • Alternative names

    • CARD2 antibody
    • CK 8 antibody
    • CK-8 antibody
    • CK8 antibody
    • CYK8 antibody
    • CYKER antibody
    • Cytokeratin endo A antibody
    • Cytokeratin-8 antibody
    • DreK8 antibody
    • EndoA antibody
    • K2C8 antibody
    • K2C8_HUMAN antibody
    • K8 antibody
    • Keratin 8 antibody
    • Keratin type II cytoskeletal 8 antibody
    • Keratin, type II cytoskeletal 8 antibody
    • Keratin-8 antibody
    • KO antibody
    • Krt 2.8 antibody
    • KRT8 antibody
    • MGC118110 antibody
    • MGC174782 antibody
    • MGC53564 antibody
    • MGC85764 antibody
    • sb:cb186 antibody
    • Type-II keratin Kb8 antibody
    see all

Images

  • 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.
  • 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).
  • 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.

    See Abreview

  • 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 (KD)
    Learn more about KD

    Click here to learn more about KD

References

This product has been referenced in:

  • Parfitt GJ Immunofluorescence Tomography: High-resolution 3-D reconstruction by serial-sectioning of methacrylate embedded tissues and alignment of 2-D immunofluorescence images. Sci Rep 9:1992 (2019). Read more (PubMed: 30760855) »
  • Hu T  et al. Developing a Novel and Convenient Model for Investigating Sweat Gland Morphogenesis from Epidermal Stem Cells. Stem Cells Int 2019:4254759 (2019). Read more (PubMed: 30863451) »
See all 45 Publications for this product

Customer reviews and Q&As

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1-7 of 7 Abreviews

Application
Western blot
Sample
Human Cell lysate - whole cell (CWR-R1 Prostate cancer cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
CWR-R1 Prostate cancer cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jul 05 2019

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (PC3 Prostate cancer cells)
Permeabilization
Yes - 0.1% Triton X-100
Specification
PC3 Prostate cancer cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde

Dr. Dimitra Kalamida

Verified customer

Submitted Mar 22 2019

Application
IHC - Wholemount
Sample
Mouse Tissue (Mammary Gland from a WT mouse)
Specification
Mammary Gland from a WT mouse

Dr. Francesco Elia Marino

Verified customer

Submitted Oct 03 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Prostate & Urethra)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate, pH 6
Permeabilization
No
Specification
Prostate & Urethra
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 01 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Bos taurus Tissue sections (Oviduct, epithelial cells)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris-EDTA, pH9.0
Permeabilization
No
Specification
Oviduct, epithelial cells
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 2.5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 27 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Sample
Mouse Cell (Differentiated P19 cell line)
Specification
Differentiated P19 cell line
Permeabilization
Yes - Triton X-100
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 24 2014

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Liver)
Specification
Liver
Fixative
Formalin
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer
Permeabilization
No
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Mar 04 2013

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