Overview

  • Product name
    Anti-Cytokeratin 8 antibody [M20]
    See all Cytokeratin 8 primary antibodies
  • Description
    Mouse monoclonal [M20] to Cytokeratin 8
  • Tested applications
    Suitable for: Flow Cyt, IHC-P, ICC, IHC-Fr, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Rat, Rabbit, Human
  • Immunogen

    Keratin isolated from the human breast carcinoma cell line MCF-7.

  • Positive control
    • Human colon for IHC-Fr
  • General notes


    Cytokeratins are a subfamily of intermediate filament proteins and are characterized by a remarkable biochemical diversity, represented in epithelial tissues by at least 20 different polypeptides. They range in molecular weight between 40 kDa and 68 kDa and isoelectric pH between 4.9 – 7.8. The individual cytokeratin polypeptides are numbered 1 to 20. The various epithelia in the human body usually express cytokeratins which are not only characteristic of the type of epithelium, but also related to the degree of maturation or differentiation within an epithelium. Cytokeratin subtype expression patterns are used to an increasing extent in the distinction of different types of epithelial malignancies. The cytokeratin antibodies are not only of assistance in the differential diagnosis of tumors using immunohistochemistry on tissue sections, but are also a useful tool in cytopathology and flow cytometric assays.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C.
  • Storage buffer
    Preservative: 0.07% Sodium azide
    Constituents: PBS, 1% Fetal calf serum
  • Concentration information loading...
  • Purity
    Protein G purified
  • Primary antibody notes
    Cytokeratins are a subfamily of intermediate filament proteins and are characterized by a remarkable biochemical diversity, represented in epithelial tissues by at least 20 different polypeptides. They range in molecular weight between 40 kDa and 68 kDa and isoelectric pH between 4.9 – 7.8. The individual cytokeratin polypeptides are numbered 1 to 20. The various epithelia in the human body usually express cytokeratins which are not only characteristic of the type of epithelium, but also related to the degree of maturation or differentiation within an epithelium. Cytokeratin subtype expression patterns are used to an increasing extent in the distinction of different types of epithelial malignancies. The cytokeratin antibodies are not only of assistance in the differential diagnosis of tumors using immunohistochemistry on tissue sections, but are also a useful tool in cytopathology and flow cytometric assays.
  • Clonality
    Monoclonal
  • Clone number
    M20
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab9023 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
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-P Use a concentration of 4 µg/ml.
ICC Use at an assay dependent concentration.
IHC-Fr 1/100 - 1/200. For human colon 20min ice cold acetone fixation was carried out with 60min incubation with ab9023 at 37C.
WB 1/100 - 1/1000.
ICC/IF 1/100.

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

  • ab9023 staining Cytokeratin 8 in Human MCF-7 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 and blocked with 5% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/100) at 4°C. An Alexa Fluor® 488-conjugated Goat anti-mouse IgG polyclonal was used as the secondary antibody (1/1000).

    See Abreview

  • ab9023 staining MCF-7 breast cancer cells, grown in 8 well chamber slides, by ICC/IF.  Cells were fixed with Acetone for 20 minutes at -20°C and permabilized with 0.1% Triton X-100 for 5 minutes at 4°C prior to blocking in 1% fish gelatin for 1 hour at RT.  The primary antibody was diluted 1/100 and incubated with the sample for 1 hour.  An Alexa Fluor® 488 conjugated donkey anti-mouse antibody diluted 1/1000 was used as the secondary.  Slides were mounted using mounting medium containing DAPI.

    See Abreview

  • The image shows different concentration of total protein from normal and tumour colon tissues.

    This picture was kindly supplied as part of the review submitted by Semona Rupchand.

    The image shows different concentration of total protein from normal and tumour colon tissues.

    This picture was kindly supplied as part of the review submitted by Semona Rupchand.

  • All lanes : Anti-Cytokeratin 8 antibody [M20] (ab9023) at 1/2000 dilution

    Lane 1 : Lysate prepared from human prostate cancer LNCaP cells
    Lane 2 : Lysate prepared from human prostate cancer PC3 cells

    Lysates/proteins at 50 µg per lane.

    Secondary
    HRP-conjugated goat polyclonal to mouse IgG at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Observed band size : 50 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 26 kDa,65 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 4 minutes

    This image is a courtesy of Anonymous Abreview

    See Abreview

  • Immunohistochemistry on paraffin section of human colon
  • Ab9023 staining human normal liver parenchima tissue. Staining is localized to cell membrane.
    Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Overlay histogram showing MCF-7 cells stained with ab9023 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.5% PBS-Tween 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 (ab9023, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/250 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.5% PBS-Tween used under the same conditions.

  • Ab9023 staining cells from a human pancreatic cancer cell line by ICC/IF.  The cells were PFA fixed and permeabilized in 0.1% Triton X-100 prior to blocking in 1% BSA for 30 minutes at 22°C.  Ab9023 was diluted 1/100 and incubated with the sample for 1 hour at 22°C.  An Alexa Fluor® 488 conjugated donkey anti-mouse antibody, diluted 1/1000, was used as the secondary.

    See Abreview

  • ab9023 staining Cytokeratin 8 in human epithelial ovarian cancer (EOC) cell line OV-MZ-6 by Immunocytochemistry/ Immunofluorescence.
    Samples were fixed with 4% PFA in PBS pH 7.4 and then permeabilised using 0.2% saponin for 30 minutes. A blocking step was performed using 1% BSA/PBS for 1 hour. Samples were then incubated with ab9023 at a 1/100 dilution in 1% BSA/PBS for 1 hour. The secondary antibody was a goat anti-mouse Alexa 488 (green) diluted 1/1000, 1% BSA/PBS for 1 hour. Samples were then incubated with phalloidin (red for actin staining) in 1% BSA/PBS for 45 minutes and counterstained with DAPI (blue for nuclei staining) in PBS for 45 minutes.
  • Immunohistochemistry on frozen section of human colon

References

This product has been referenced in:
  • Belvedere R  et al. Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway. Sci Rep 6:29660 (2016). WB . Read more (PubMed: 27412958) »
  • McCormick NH  et al. ZnT4 (SLC30A4)-null ("lethal milk") mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation. Am J Physiol Regul Integr Comp Physiol 310:R33-40 (2016). WB ; Mouse . Read more (PubMed: 26538236) »

See all 18 Publications for this product

Customer reviews and Q&As

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (MCF-7 Cells)
Permeabilization
Yes - 0.25% Triton X-100
Specification
MCF-7 Cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jul 22 2015

Thank you for contacting us. I am sorry that the ab14053 is currently out of stock. Having looked into the suitability of ab9023 as a replacement I would now not recommend its use. Although some of our customers have used it successfully in mouse, ...

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (skin)
Specification
skin
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH6
Permeabilization
No
Blocking step
Vector MOM Kit Mouse IgG as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3.6% · Temperature: RT°C
Username

Mr. Manoj Kumar Valluru

Verified customer

Submitted May 24 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (prostate)
Specification
prostate
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.2% Triton-X
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Oct 21 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (prostate tumor cell line)
Loading amount
50 µg
Specification
prostate tumor cell line
Gel Running Conditions
Reduced Denaturing (10% Tris-gly)
Blocking step
BSA as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Oct 21 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Loading amount
20 µg
Specification
HeLa
Gel Running Conditions
Reduced Denaturing (Bis-Tris 4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Aug 01 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (pancreatic cancer cell line)
Loading amount
20 µg
Specification
pancreatic cancer cell line
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5µg/mL · Temperature: 22°C
Username

Dr. Hannah Whiteman

Verified customer

Submitted Jul 10 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (pancreatic cancer cell lines)
Specification
pancreatic cancer cell lines
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 22°C
Username

Dr. Hannah Whiteman

Verified customer

Submitted Jun 18 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (MCF-7 breast cancer cells)
Specification
MCF-7 breast cancer cells
Fixative
Acetone
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
Fish Gelatin as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Mar 14 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Colon cancer cell lines HCT116/HT29/Caco2)
Loading amount
40 µg
Specification
Colon cancer cell lines HCT116/HT29/Caco2
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% in TBST
Username

Abcam user community

Verified customer

Submitted Sep 18 2006

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