Overview

  • Product name

    Anti-Cytokeratin 8 antibody [M20]
    See all Cytokeratin 8 primary antibodies
  • Description

    Mouse monoclonal [M20] to Cytokeratin 8
  • Host species

    Mouse
  • 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
  • Purity

    Tissue culture supernatant
  • 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

  • Lane 1: A431 cell lysate (20 µg)

    Lane 2: MCF7 cell lysate (20 µg)

    Lane 3: HeLa wildtype cell lysate (20 µg)

    Lane 4: KRT8 HeLa knockout cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab9023 observed at 54 kDa. Red - loading control, ab181602 observed at 37 kDa.

    ab9023 was shown to react with Cytokeratin 8 in HeLa wildtype. Loss of signal was observed when knockout sample ab263785 was used. Wild-type and Cytokeratin 8 knockout samples were subjected to SDS-PAGE. ab9023 and Anti-GAPDH antibody EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 100 - 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
  • 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
    All lanes : 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

    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:

  • Ehsani E  et al. Improved differentiation of human enriched CD133+CD24+ renal progenitor cells derived from embryonic stem cell with embryonic mouse kidney-derived mesenchymal stem cells co-culture. Differentiation 109:1-8 (2019). Read more (PubMed: 31323479) »
  • Nguyen QH  et al. Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity. Nat Commun 9:2028 (2018). Read more (PubMed: 29795293) »
See all 22 Publications for this product

Customer reviews and Q&As

1-4 of 4 Q&A

Question
Answer

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, we have also had several reports of it producing non-specific staining. I would therefore suggest and antibody such as ab59400 which has been tested with mouse samples and with ELISA, immunocytochemistry, immunohistochemistry and westernblotting. This is a rabbit polyclonal and is currently in stock. This antibody is covered by the Abpromise for the application and species you intend to use, it would therefore not qualify for a testing discount. This does however mean that if you are not satisfied with the results and we are not able to help you improve them, you would be eligable for a free replacement (or your choice) or a refund. If there is an alternative antibody that you prefer which has not been tested in mouse or for your application then I would be able to issue the testing discount.  If you feel the replacement suggested is not suitable replacement please could you give me the full details of what experiments you intend to do and I can see which antibody may match your criteria.

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Answer

Thank you for your enquiry. We have a chicken polyclonal to Cytokeratin 8, catalog number ab14053, that works in mouse tissue and in Western blot. Please find the datasheet at the following link: https://www.abcam.com/index.html?datasheet=14053 I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for your enquiry. The originator of ab9023 has passed along the following FACS protocol to use with this antibody. Example protocol: anti-cytokeratin M20 FACS analysis Materials: •Methanol -20 °C •PBS •PBS/BSA (1 mg/ml BSA in PBS) •Propidium/RNAse solution (20 µg/ml propidium iodide, 100 µg/ml RNAse in PBS) Antisera: Primary antibody •M20 (mouse anti-cytokeratin 8), 1:20 in PBS/BSA Secondary antibody •FITC conjugated anti-mouse Ig (DAKO F313 1:15 in PBS/BSA) Cell harvesting and fixation: •Harvest cells quantitatively by trypsinisation •Spin cell suspension for 5 min at 400 x g •Resuspend cell pellet in 200 µl PBS •Add under constant agitation 3.5 ml cold methanol (-20 °C) •Store cell suspension in Methanol in refrigerator until use Immunocytochemistry: •Spin methanol fixed cell suspension for 5 min at 400 x g •Discard supernatant and resuspend pellet in 1 ml culture medium •Spin cells at 400 x g •Incubate approximately 10^6 cells in 100 µl with the primary antibody for 1h at RT or overnight at 4 °C •Rinse twice with PBS/BSA •Incubate cells in 100 µl with FITC conjugated secondary antibody for 1 h at RT •Rinse with PBS/BSA •Resuspend cells in propidium iodide/RNAse solution Incubate for 15 min on ice prior to flow cytometry analysis

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Answer

Thank you for your phone call. Regarding ab2530, ab2531, and ab8477, the epitopes have not been determined. Once I have heard back from the originator of ab9023 I will let you know.

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