Overview

  • Product name

  • Description

    Rabbit polyclonal to DCAMKL1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, Flow Cyt, ELISA, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human DCAMKL1 aa 690-720 (C terminal) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
    (Peptide available as ab110799)

  • Positive control

    • WB: Mouse heart and human brain tissue lysates. IHC-P: Human hepatocarcinoma tissue. ICC/IF: MCF7 cells.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.09% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Ammonium Sulphate Precipitation
  • Purification notes

    This antibody is prepared by Saturated Ammonium Sulfate (SAS) precipitation followed by dialysis against PBS.
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab37994 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
ICC/IF Use a concentration of 1 µg/ml.
Flow Cyt 1/33.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

ELISA 1/100.
WB 1/1000. Detects a band of approximately 82 kDa (predicted molecular weight: 82 kDa).
IHC-P 1/10 - 1/50.

Target

  • Function

    Probable kinase that may be involved in a calcium-signaling pathway controlling neuronal migration in the developing brain. May also participate in functions of the mature nervous system.
  • Tissue specificity

    In fetal tissues, highly expressed in brain, detectable in lung and liver, but not in kidney. In adult tissues, expressed ubiquitously in the brain, detectable in the heart, liver, spleen, thymus, prostate, testis, ovary, small intestine and colon. The type A isoforms seem to be expressed predominantly in fetal brain whereas type B isoforms are expressed abundantly in both fetal and adult brain.
  • Sequence similarities

    Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. CaMK subfamily.
    Contains 2 doublecortin domains.
    Contains 1 protein kinase domain.
  • Information by UniProt
  • Database links

  • Alternative names

    • Calcium/calmodulin-dependent protein kinase type I-like CPG16 antibody
    • CL1 antibody
    • CLICK1 antibody
    • Cpg16 antibody
    • DCDC3A antibody
    • Dcl antibody
    • Dclk antibody
    • Dclk1 antibody
    • DCLK1_HUMAN antibody
    • Doublecortin domain-containing protein 3A antibody
    • Doublecortin-like and CAM kinase-like 1 antibody
    • Doublecortin-like kinase 1 antibody
    • KIAA0369 antibody
    • Serine/threonine-protein kinase DCAMKL1 antibody
    • Serine/threonine-protein kinase DCLK1 antibody
    see all

Images

  • Immunohistochemical analysis of paraffin-embedded normal mouse small intestine tissue labeling DCAMKL1 with ab37994 at 1/10 dilution

  • Anti-DCAMKL1 antibody (ab37994) at 1/1000 dilution + Mouse heart tissue lysate at 35 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/5000 dilution

    Predicted band size: 82 kDa
    Observed band size: 82 kDa
    Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.



    Incubation time was overnight at 4°C. Blocking/Dilution buffer: 5% NFDM/TBST.

  • ICC/IF image of ab37994 stained MCF7 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 (ab37994, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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-DCAMKL1 antibody (ab37994) at 1/750 dilution (for 20 hours at 4°C) + Human brain whole tissue lysate at 22 µg

    Secondary
    An undiluted HRP-conjugated Goat anti-rabbit polyclonal

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 82 kDa
    Observed band size: 82 kDa


    Exposure time: 3 minutes


    Blocking step: 5% Milk for 1 hour at 18C°C

    See Abreview

  • All lanes : Anti-DCAMKL1 antibody (ab37994) at 1/8000 dilution

    Lane 1 : Human brain lysate
    Lane 2 : Mouse brain lysate
    Lane 3 : Rat brain lysate
    Lane 4 : TT whole lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution

    Predicted band size: 82 kDa

References

This product has been referenced in:

  • Cheng X  et al. A novel serotonin-containing tuft cell subpopulation in mouse intestine. Cell Tissue Res N/A:N/A (2019). Read more (PubMed: 30666535) »
  • Khodadadi Kohlan A  et al. Induction of let-7e gene expression attenuates oncogenic phenotype in HCT-116 colorectal cancer cells through targeting of DCLK1 regulation. Life Sci 228:221-227 (2019). Read more (PubMed: 31075231) »
See all 25 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Colon tissue)
Permeabilization
Yes - 0.1% Triton X -100
Specification
Colon tissue
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Apr 29 2019

Application
IHC - Wholemount
Sample
Mouse Tissue (trachea)
Specification
trachea

Saltanat Ualiyeva

Verified customer

Submitted Dec 11 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (intestine)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citric buffer
Permeabilization
No
Specification
intestine
Blocking step
H2O2 as blocking agent for 10 minute(s) · Concentration: 3% · Temperature: 20°C
Fixative
Paraformaldehyde

Miss. Xiaowen Cheng

Verified customer

Submitted Jul 04 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mM sodium citrate pH6
Sample
Mouse Tissue sections (Intestine)
Specification
Intestine
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 16 2014

Question
Answer

Thank you for your phone call.

A suitable isotype control for ab31704 and ab37994 would be: ab27478

https://www.abcam.com/Rabbit-polyclonal-IgG-ab27478.html


Please find herethe discount code and more detailed information for trying ab31704 in flow cytometry:

DISCOUNT CODE: xxx
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab31704 in flow cytometry. This code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for flow cytometryand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Read More

Answer

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 for one vial of ab31704.

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

Please note that in general we do not stock small sample sizes of our products so I can't send a small amout to test of ab37994.

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

Answer

Thanks for your email and for the detailed description of the problem.

You have done a good job in attempts to optimize the protocol. This item is purchased relativelyfrequently and there has only been 1 other recent complaint (within the last year). Nonetheless, it appears the vial of ab37994 is not working as expected. If you have purchased this product within the last 6 months or so I would be happy to offer a replacement, credit or refund.

In your reply, please include your PO number or Abcam order reference number so I can process your request.

Read More

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

Question
Answer

Thank you for contacting us. We know that the antibody recognizes the 2 isoforms: AL, BL (which corresponds to isoforms 2 and 4) due to sequence homology. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Human Intestine)
Specification
Human Intestine
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C

Abcam user community

Verified customer

Submitted Sep 01 2010

1-10 of 13 Abreviews or Q&A

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