Overview

  • Product name
    Anti-Cytokeratin 19 antibody
    See all Cytokeratin 19 primary antibodies
  • Description
    Rabbit polyclonal to Cytokeratin 19
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC - Wholemount, WB, IHC-P, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human Cytokeratin 19 aa 300 to the C-terminus. The exact sequence is proprietary.
    Database link: P08727

  • Positive control
    • IHC-P: Human skin and colon tissue; Mouse skin tissue. WB: HepG2 and MDA-MB-361 cell lysates.

Properties

Applications

Our Abpromise guarantee covers the use of ab15463 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
IHC - Wholemount Use at an assay dependent concentration. Used at a dilution of 1/200 for 24 hr on mouse whole mounts of tail skin (see Abreview submitted by Jesus Espada).
WB Use a concentration of 1 µg/ml. Detects a band of approximately 45 kDa (predicted molecular weight: 40 kDa).
IHC-P 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Boil in citrate buffer for 10 minutes, cool at room temperature for 20 minutes. After blocking, incubate primary antibody 1:100 for 10 minutes at room temperature.

IHC-Fr 1/50.

Target

  • Function
    Involved in the organization of myofibers. Together with KRT8, helps to link the contractile apparatus to dystrophin at the costameres of striated muscle.
  • Tissue specificity
    Expressed in a defined zone of basal keratinocytes in the deep outer root sheath of hair follicles. Also observed in sweat gland and mammary gland ductal and secretory cells, bile ducts, gastrointestinal tract, bladder urothelium, oral epithelia, esophagus, ectocervical epithelium (at protein level). Expressed in epidermal basal cells, in nipple epidermis and a defined region of the hair follicle. Also seen in a subset of vascular wall cells in both the veins and artery of human umbilical cord, and in umbilical cord vascular smooth muscle. Observed in muscle fibers accumulating in the costameres of myoplasm at the sarcolemma in structures that contain dystrophin and spectrin.
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Developmental stage
    Present in hair follicles at all stages of development.
  • Domain
    This keratin differs from all other IF proteins in lacking the C-terminal tail domain.
  • Information by UniProt
  • Database links
  • Alternative names
    • 40 kDa keratin intermediate filament antibody
    • CK 19 antibody
    • CK-19 antibody
    • CK19 antibody
    • Cytokeratin 19 antibody
    • Cytokeratin-19 antibody
    • K19 antibody
    • K1C19_HUMAN antibody
    • K1CS antibody
    • Keratin 19 antibody
    • Keratin type I 40 kD antibody
    • Keratin type I 40kD antibody
    • Keratin type I cytoskeletal 19 antibody
    • Keratin, type I cytoskeletal 19 antibody
    • Keratin, type I, 40 kd antibody
    • Keratin-19 antibody
    • KRT19 antibody
    • MGC15366 antibody
    see all

Images

  • Human skin stained with anti-Cytokeratin 19 antibody (ab15463).
  • All lanes : Anti-Cytokeratin 19 antibody (ab15463) at 1 µg/ml

    Lane 1 : Human placenta tissue lysate - total protein (ab29745)
    Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 3 : MDA-MB-361 (Human breast adenocarcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Predicted band size: 40 kDa
    Observed band size: 45 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 75 kDa. We are unsure as to the identity of these extra bands.

  • ab15463 staining Cytokeratin 19 in human colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500 in blocking buffer) for 16 hours at 22°C. A Biotin-conjugated Goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse developing skin tissue labeling Cytokeratin 19 with ab15463 at 1/500 dilution. The tissue was fixed with formaldehyde; heat-mediated antigen retrieval was performed using a citric acid. Tissue was blocked with 2% BSA for 10 minutes at 21°C. A polyclonal goat anti-rabbit IgG biotin conjugated antibody was used ar 1/300 dilution.  Image shows strong immunopositivity in the cytoplasm of basal cells of the epidermis and a moderate hair follicle cell positivity.

    See Abreview

  • This image shows formalin-fixed mouse skin stained with Cytokeratin 19 antibody (1/50 dilution) after pepsin digestion.
  • ab15463 staining Cytokeratin 19 in the Mouse skin tissue sections by IHC-Fr (Frozen Sections). Tissue samples were fixed with methanol and blocked with 3% Goat serum for 30 minutes at 20°C. The sample was incubated with primary antibody (1/50 in PBS) at 4°C for 14 hours. An HRP-conjugated goat polyclonal to rabbit IgG (1/100) was used as secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Bishehsari F  et al. KRAS mutation and epithelial-macrophage interplay in pancreatic neoplastic transformation. Int J Cancer 143:1994-2007 (2018). Read more (PubMed: 29756386) »
  • Chen NM  et al. Context-Dependent Epigenetic Regulation of Nuclear Factor of Activated T Cells 1 in Pancreatic Plasticity. Gastroenterology 152:1507-1520.e15 (2017). Read more (PubMed: 28188746) »
See all 37 Publications for this product

Customer reviews and Q&As

1-10 of 32 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (pancreas)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mm sodium citrate PH 6.0
Permeabilization
No
Specification
pancreas
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Apr 04 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Colon
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted Feb 13 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Developing skin)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Developing skin
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted Dec 02 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
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
BiocareMedical's Rodent Block M as blocking agent for 20 minute(s) · Concentration: 100% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Mar 05 2013

Answer

Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

This antibody hasn't yet been validated in pig, so we are unsure whether or not it will work in this species. Would you have access to run a positive control with mouse or human skin?

Is the trouble that you are not seeing any staining? If you aren't seeing any signal, you may want to cut down on the blocking percentage and time of incubation.

We have heard that epitope retrieval with citrate buffer ph 6.0 for 10 minutes at the microwave at 600 W and blocking the endogen peroxidase with 3% H2O2 in Methanol for 10 min, with primary at 1/50 overnight 4C works well. The secondary was used at 1/200. What dilution of the secondary have you tried?

Should the suggestions not improve the results, please do let me know.

I hope this information is helpful, and I thank you for your cooperation.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Liver)
Specification
Liver
Fixative
Formaldehyde
Blocking step
PBS + 0.1% Triton X-100, 1% BSA, 10% goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Mr. justin kenkel

Verified customer

Submitted Jul 27 2012

Question

Thank you for your prompt reply, please find below the conditions used for our experiments, as well as attachments of the western blots and the paper that you requested.



Kindest regards,

1) Abcam product code ab37994 and ab15463



2) Abcam order reference number or product batch number: HGA4500684274



3) Description of the problem: Both antibodies have failed to detect any protein on western blot and immunofluorescence in cell lines fully expected to produce those proteins, as shown in previously published work (please see attached paper Ali et al).



For Western blot:



4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): Whole cell lysates

Lysis buffer: EBC

Protease inhibitors: Complete protease inhibitor kit.

Phosphatase inhibitors

Reducing agent

Boiling for ≥5 min? yes

Protein loaded ug/lane or cells/lane: 10ul of cell lysate (standardized for protein concentration by Bradford assay.

Positive control: HUH7

Negative control: None



5) Percentage of gel: Protean pre-cast gel 4-20%

Type of membrane: PDVF

Protein transfer verified: yes

Blocking agent and concentration: 10% dried milk powder/TBS-tween

Blocking time: 2 hours

Blocking temperature: room temperature



6) Primary antibody (If more than one was used, describe in “additional notes”) :CK19 and DCAMKL-1

Concentration or dilution: 1:1000 for both antibodies

Diluent buffer: 5% dried milk powder/TBS-Tween

Incubation time: 2 hours

Incubation temperature: Room temperature



7) Secondary antibody:

Species: Goat

Reacts against: Rabbit

Concentration or dilution: 1:2500

Diluent buffer: 5% milk/TBS-tween

Incubation time: 2 hours

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate: HRP



8) Washing after primary and secondary antibodies:

Buffer: TBS-Tween

Number of washes: 2x quick washes, followed by 1x 10 minute wash, then 3x 5minute washes



9)Detection method: ECL



10) How many times have you run this staining? Once for Western blot and once for immunofluorescence

Do you obtain the same results every time? -

What steps have you altered to try and optimize the use of this antibody? –



Attached document shows these results, as well as confirmation of even loading (GAPDH).



For Immunofluorescence



4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): Cells grown on cover slip in 6-well plate, then treated with 4% paraformaldehyde, prior to membrane disruption with 0.2% TRITON-X100 for 5 minutes.

Positive control: HUH7

Negative control: None



5) Blocking agent and concentration: Blocking carried out with 10%FCS/PBS alongside primary and secondary antibody probing as below.



6) Primary antibody (If more than one was used, describe in “additional notes”) :CK19 and DCAMKL-1

Concentration or dilution: 1:200 (CK19) and 1:250 (DCAMKL-1).

Diluent buffer: 10%FCS/PBS

Incubation time: 1 hour

Incubation temperature: Room temperature



7) Secondary antibody:

Species: Goat

Reacts against: Rabbit

Concentration or dilution: 1:200

Diluent buffer: 10%FCS/PBS

Incubation time: 1 hour

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate: ALEXA-FLUR 594



8) Washing after primary and secondary antibodies:

Buffer: PBS

Number of washes: 3



9)Detection method: Fluorescent light microscope

10) How many times have you run this staining? Once for Western blot and once for immunofluorescence

Do you obtain the same results every time? -

What steps have you altered to try and optimize the use of this antibody? -

Read More
Answer

Thank you for taking the time to complete our questionnaire and sending me the publication. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.


Although the other antibody has worked well using this procedure, individual antibodies will often require optimization. I would like to offer some suggestions to help optimise the results from ab15463 and ab37994.

1.) I suggest to use a different positive control than HUH7 cells. As shown in figure 1 of the publication provided HUH7 cells do not show any expression of CK-19 and a only a very low expression of DCAMKL-1. I can recommend to use HepG2 cells or shin for ab15463 and MCF for ab37994 as indicated on the respective datasheets.

2.) I also can recommend to incubate primary antibodies over night at 4C. This will ensure the best saturation and specificity of the antibody.

3.) For the WB, I suggest to use less than 10% milk powder as blocking agent (maybe try a different blocking agent like BSA which can sometimes have a big impact on signal strength).

4.) For ICC/IF I can recommend to try a milder permeabilisation agent./ For example 0.1 Tween in the blocking buffer for 1 hour.


I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Mammary Gland duct)
Specification
Mammary Gland duct
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Mar 20 2012

Answer

Thank you for contacting us.

I am sorry to hear that the antibody is giving you problems.

As we discussed over the phone, please let me know what staining patternthe MCF7 cells give and we can discuss further how to proceed.

I wish you good luck andlook forward to hear back from you.

Read More

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx with ab20206.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

1-10 of 32 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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