Overview

  • Product name

    Anti-Cytokeratin 18 antibody [CK-18]
    See all Cytokeratin 18 primary antibodies
  • Description

    Mouse monoclonal [CK-18] to Cytokeratin 18
  • Host species

    Mouse
  • Specificity

    No cross reactivity with other proteins.
  • Tested applications

    Suitable for: Flow Cyt, WB, IHC-P, ICC/IF, IHC-Fr, IPmore details
  • Species reactivity

    Reacts with: Rat, Human
  • Immunogen

    Tissue, cells or virus corresponding to Human Cytokeratin 18. Human epidermal carcinoma A431 and MCF7 human breast cancer cell lines.

  • Positive control

    • This antibody gave a positive signal in HCT116 cell line in IF/ICC.

Properties

Applications

Our Abpromise guarantee covers the use of ab82254 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 1µg for 106 cells.

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

WB Use a concentration of 0.5 - 1 µg/ml. Predicted molecular weight: 48 kDa.
IHC-P Use a concentration of 1 - 2 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.
IHC-Fr Use a concentration of 0.5 - 2 µg/ml. Use formalin or acetone fixed tissues.
IP Use a concentration of 5 µg/ml.

Target

  • Function

    Involved in the uptake of thrombin-antithrombin complexes by hepatic cells (By similarity). When phosphorylated, plays a role in filament reorganization. Involved in the delivery of mutated CFTR to the plasma membrane. Together with KRT8, is involved in interleukin-6 (IL-6)-mediated barrier protection.
  • Tissue specificity

    Expressed in colon, placenta, liver and very weakly in exocervix. Increased expression observed in lymph nodes of breast carcinoma.
  • Involvement in disease

    Defects in KRT18 are a cause of cirrhosis (CIRRH) [MIM:215600].
  • Sequence similarities

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

    Phosphorylation at Ser-34 increases during mitosis. Hyperphosphorylated at Ser-53 in diseased cirrhosis liver. Phosphorylation increases by IL-6.
    Proteolytically cleaved by caspases during epithelial cell apoptosis. Cleavage occurs at Asp-238 by either caspase-3, caspase-6 or caspase-7.
    O-glycosylated at multiple sites; glycans consist of single N-acetylglucosamine residues.
  • Cellular localization

    Cytoplasm > perinuclear region.
  • Information by UniProt
  • Database links

  • Alternative names

    • Cell proliferation inducing gene 46 protein antibody
    • Cell proliferation inducing protein 46 antibody
    • Cell proliferation-inducing gene 46 protein antibody
    • CK 18 antibody
    • CK-18 antibody
    • CK18 antibody
    • CYK 18 antibody
    • CYK18 antibody
    • Cytokeratin 18 antibody
    • Cytokeratin endo B antibody
    • Cytokeratin-18 antibody
    • K 18 antibody
    • K18 antibody
    • K1C18_HUMAN antibody
    • KA18 antibody
    • Keratin 18 antibody
    • Keratin 18, type I antibody
    • Keratin D antibody
    • keratin, type I cytoskeletal 18 antibody
    • Keratin-18 antibody
    • Krt18 antibody
    see all

Images

  • ICC/IF image of ab82254 stained HCT116 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab82254, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  • Anti-Cytokeratin 18 antibody [CK-18] (ab82254) + MCF-7 whole cell lysate

    Predicted band size: 48 kDa

  • ab82254 staining Cytokeratin 18 in paraffin-embedded human rectal cancer tissue.
  • Overlay histogram showing MCF7 cells stained with ab82254 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% 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 (ab82254, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 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 MCF7 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Cytokeratin 18 was immunoprecipitated using 0.5mg MCF7 Whole cell lysate, 5µg of Mouse monoclonal to Cytokeratin 18 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, MCF7 Whole cell lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab82254.
    Secondary: Protein G-HRP at 1/500 dilution.
    Band: 45kDa; Cytokeratin 18: non specific bands - 49kDa: We are unsure as to the identity of this extra band.

References

This product has been referenced in:

  • Puig P  et al. Multiple immunofluorescence assay identifies upregulation of Active ß-catenin in prostate cancer. BMC Res Notes 12:68 (2019). Read more (PubMed: 30700322) »
  • Huang TW  et al. Chitosan-hyaluronan: promotion of mucociliary differentiation of respiratory epithelial cells and development of olfactory receptor neurons. Artif Cells Nanomed Biotechnol 47:564-570 (2019). Read more (PubMed: 30857434) »
See all 21 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab82254. I would also appreciate if you can confirm some further details: Before the protein blocking step we suggest use of a permeablization step. This will allow antibody access to the antigen which has perinuclear localization. This can be accomplished by incubation with PBS or TBS containing 0.25% Triton X-100 (or 100uM digitonin or 0.5% saponin) for 10 minutes followed with 3 x 5 minute PBS washes. Proceed as normal after these steps. An alternative is to fix the cells with 100% methanol or 100% acetone. These fixatives will also permeablize the cells. Could you please specify the species and cell line used in these experiments? Has the recommended positive control for this product (HCT116 cells) been used? Were results the same?

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Answer

Thank you for your response.

It is important to mention that only ab133263 has been tested for IHC on formalin-fixed tissue samples. The other product (ab82254)is suitable for frozen sections, not been tested for formalin-fixed tissue samples.

I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More

Answer

Thank you for your enquiry and your interest in our products.

Here are a few useful and interesting websites, you may wish to read through before starting the experiments:
https://docs.abcam.com/pdf/immunology/fluorochromes_table_updated.pdf
https://www.abcam.com/index.html?pageconfig=resource&rid=12922
https://www.abcam.com/index.html?pageconfig=popular_protocols
Both antibodies are suitable for detecting rat cytokeratin 18 and have been tested for immunohistochemistry. You may need to consider how you wish to fix the samples i.e. formalin, acetone, methanol, ethanol etc.









These antibodies are unconjugated, so you need a compatible secondary antibody which is conjugated for the detection. In order to be able to select the right secondary antibody, it is essential to understand the isotype and the host species of your chosen primary antibody:

- ab82254: mouse IgG1 so the secondary should be anti-mouse IgG1;

- ab133263: rabbit IgG1 so the secondary should be anti-rabbit IgG1;

I wouldrecommend conducting a search for the secondary antibody using the advance search engine. You can select the target species, the host species, isotype, format, conjugate etc:

https://www.abcam.com/index.html?pageconfig=productmap&cl=918

I hope this helps and if I can assist further, please do not hesitate to contact me.

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