Overview

  • Product name
  • Description
    Rabbit polyclonal to Dnmt3L
  • Host species
    Rabbit
  • Specificity
    This antibody is expected to cross-react with mouse as the sequence of the human immunogen shows 76% identity with mouse.
  • Tested applications
    Suitable for: ICC/IF, ELISA, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Cow, Human
  • Immunogen

    Synthetic peptide corresponding to Human Dnmt3L aa 152-164 conjugated to keyhole limpet haemocyanin.
    Sequence:

    C-GLLQRRRKWRSQL


    Database link: Q9UJW3

  • General notes

    DNMT 3L is a nuclear protein which has similarity to DNA methyltransferases, involved in de novo methylation of CpG islands. CpG methylation is an epigenetic modification that is important for embryonic development, imprinting, and X-chromosome inactivation. Studies in mice have demonstrated that DNA methylation is required for mammalian development. This gene encodes a nuclear protein with similarity to DNA methyltransferases. This protein is not thought to function as a DNA methyltransferase as it does not contain the amino acid residues necessary for methyltransferase activity. However, this protein does stimulate de novo methylation by DNA cytosine methyltransferase 3 alpha and it is thought to be required for the establishment of maternal genomic imprints. This protein also mediates transcriptional repression through interaction with histone deacetylase 1.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 0.42% Potassium phosphate, 0.87% Sodium chloride
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    This is an affinity purified antibody produced by immunoaffinity chromatography using the immunizing peptide after immobilization to a solid phase. This antibody is expected to cross-react with mouse as the sequence of the human immunogen shows 76% identity with mouse. Reactivity with DNMT3L from other species has not yet been tested.
  • Primary antibody notes
    DNMT 3L is a nuclear protein which has similarity to DNA methyltransferases, involved in de novo methylation of CpG islands. CpG methylation is an epigenetic modification that is important for embryonic development, imprinting, and X-chromosome inactivation. Studies in mice have demonstrated that DNA methylation is required for mammalian development. This gene encodes a nuclear protein with similarity to DNA methyltransferases. This protein is not thought to function as a DNA methyltransferase as it does not contain the amino acid residues necessary for methyltransferase activity. However, this protein does stimulate de novo methylation by DNA cytosine methyltransferase 3 alpha and it is thought to be required for the establishment of maternal genomic imprints. This protein also mediates transcriptional repression through interaction with histone deacetylase 1.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab3493 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 1/500.
ELISA 1/1000 - 1/3000. 1/1000 - 1/3000 against 0.1µg of the immunizing peptide.
WB Use a concentration of 0.4 µg/ml. Predicted molecular weight: 43.6 kDa. Use at an assay dependent dilution. The target may need to be immunoprecipitated to identify the target in WB. Predicted molecular weight: 43.6 kDa.
IP 1/100.

Target

  • Function
    Catalytically inactive regulatory factor of DNA methyltransferases. It is essential for the function of DNMT3A and DNMT3B. Activates DNMT3A and DNMT3B by binding to their catalytic domain. Accelerates the binding of DNA and AdoMet to the methyltransferases and dissociates from the complex after DNA binding to the methyltransferases. Recognizes unmethylated histone H3 lysine 4 (H3K4) and induces de novo DNA methylation by recruitment or activation of DNMT3.
  • Tissue specificity
    Expressed at low levels in several tissues including testis, ovary, and thymus.
  • Sequence similarities
    Belongs to the C5-methyltransferase family.
    Contains 1 ADD-type zinc finger.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Cytosine 5 methyltransferase 3 like protein antibody
    • DNA (cytosine 5 ) methyltransferase 3 like antibody
    • DNA (cytosine-5)-methyltransferase 3-like antibody
    • DNA cytosine 5 methyltransferase 3 like protein antibody
    • DNA methyltransferase 3 like protein antibody
    • DNM3L_HUMAN antibody
    • Dnmt 3L antibody
    • Dnmt3l antibody
    • Human cytosine 5 methyltransferase 3 like protein antibody
    • MGC1090 antibody
    see all

Images

  • Transfection of U2OS cells (10cm dish) using 3µg of GAL4-DNMT3L. Protein extraction using IPH 150mM. IP and WB conditions, see Fuks et al. (2000). Nature Genetics 24: 88-91.WB using 0.4µg/ml in TBS milk 2%, BSA 0.5%. Dectection using ECL.

    Note: ab3493 worked in WB following IP, but did not work in a straight WB.

  • Anti-Dnmt3L antibody (ab3493) at 1/1000 dilution + Rat C6 cells, whole cell lysate at 25 µg

    Secondary
    HRP conjugated goat anti-rabbit antibody at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 43.6 kDa
    Observed band size: 52 kDa
    why is the actual band size different from the predicted?


    Exposure time: 30 seconds

    See Abreview

  • Immunofluorescence analysis of 2-cell mouse embryos, staining Dnmt3L with ab3493.

    Embryos were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 0.5% blocking reagent in TNT (0.1 M, Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween-20) buffer for 20 min at 4°C. Embryos were incaubted with primary antibody (1/500) for 1 hour at 37°C. An AlexaFluor®488-conjugated goat anti-rabbit IgG (1/400) was used as the secondary antibody.

References

This product has been referenced in:
  • Peng J  et al. The detrimental effects of glucocorticoids exposure during pregnancy on offspring's cardiac functions mediated by hypermethylation of bone morphogenetic protein-4. Cell Death Dis 9:834 (2018). Read more (PubMed: 30082698) »
  • Nanan KK  et al. Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource. Nucleic Acids Res 45:12780-12797 (2017). Read more (PubMed: 29244186) »
See all 10 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Answer

Thank you for your inquiry.
I can confirm that it was not experimentally tested if this antibody shows any cross-reaction with the proteins DNMT3A and/or DNMT3B.
After performing a short BLAST search with the antigen, it is very unlikely that the antibody cross-reacts.
The BLAST search did not bring up these proteins in any species, but only showed DNMT3L proteins from different species.
I hope this information is helpful and wish you good luck with your research.

Read More

Answer

Thank you for your note.

This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab3493. For your information, the internal reference note for this credit is CN20018. You can use this credit in the near future for any of the products which are in the catalogue.

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

Read More

Answer

Thank you for your response and for sending some additional information and images. Your co-operation is very much appreciated.

I could offer your customer either a new vial as a free of charge replacement vial or a credit note which can be used in future purpose.

Please could you discuss with your customer and let me know how to proceed with this case.

I look forward to hearing from you soon.

Read More

Question

Dear technical support team:

This customer has purchased ab3493 (Anti-Dnmt3L antibody) and has conducted the WB several times with mouse sample. The results show high background and non-specific signal; therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her result?

I attached the image in this letter and her experiment step as follow:

1. Order details:


Batch number: GR24271-3

Po: 1002399

Abcam product code: ab3493

Antibody storage conditions (temperature/reconstitution etc): -20C




2. Please describe the problem (high background, wrong band size, more bands, no band etc).

High background, many non-specific band, No target band

3. On what material are you testing the antibody in WB?

· Species: mouse

· What’s cell line or tissue: germ cell, ES cells, MEF, 3T3

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: Recombinant protein



3. The lysate


How much protein was loaded: 20 ug

What lysis buffer was used: RIPA



What protease inhibitors were used: Fermentas ProteoBlock

What loading buffer was used: LONZA ProSieve ProTrack

Phosphatase inhibitors: NO

Did you heat the samples: temperature and time: YES, 95C, 3 mins




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: non reducing gel

Reducing agent:

Gel percentage : 10%

Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, transfer 1hr, 250 mA, pre-stain markers and internal control GAPDH were seen.




5. Blocking conditions


Buffer: 6% milk in PBST, or Millipore BLOK-CH, or 6% BSA

Blocking agent: milk, BSA, serum, what percentage: 6% milk or Millipore BLOK-CH, or 6% BSA, each has been used.



Incubation time: over night

Incubation temperature: 4C




6. Primary Antibody


Species: rabbit

Reacts against: Dnmt3L


· At what dilution(s) have you tested this antibody:1000x, 500x

· What dilution buffer was used: PBST, Calbiochem SignalBoost™Immunoreaction Enhancer

· Incubation time: 2 hrs

· Incubation temperature: RT

· What washing steps were done: PBST wash 30mins for 5 times



7. Secondary Antibody


Species: Goat

Reacts against: anti-rabbit IgG

At what dilution(s) have you tested this antibody: 2000X, 5000X

Incubation time: 1hr, 2hrs

Wash steps: PBST wash 30mins for 5 times

Fluorochrome or enzyme conjugate:HRP

Do you know whether the problems you are experiencing come from the secondary? NO, the secondary antibody has been used successfully for other primary antibody. We can see the non-specific bands.




8. Detection method
ECl, ECl+, other detection method: ECL



9. Did you apply positive and negative controls along with the samples? Please specify.

YES. We used wild type and Dnmt3L knockout ES cells. The target protein expression has been verified by other worked primary antibody and Q-RT-PCR.

10. Optimization attempts

· How many times have you tried the Western? For 5 times.

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):

YES. The non-specific bands were not from the secondary antibody. It’s OK seeing non-specific bands. We cannot see the differences of target band between WT and Dnmt3L KO ES cells.

· Do you obtain the same results every time e.g. are background bands always in the same place? Yes

· What steps have you altered?




Primary and secondary incubation time.

The antibody dilution.

Using the Calbiochem signal boost

Using the Millipore Blok-CH






Could you please help this customer to solve the problem?

Thanks for your kindly help.

Best regards.

Read More
Answer

Thank you for your enquiry regardingab3493 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer ishaving problems with this antibody.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Immunoprecipitation: As the datasheet indicates, ab3493 worked in WB following IP, but did not work in a straight WB. Please advise your customer to perform an IP firts.

2) Transfection and KO: As teh customer has used transfected cells lines and KO cells, it would be great if you could provide some more details:

2.1 Does the construct contain full sequence or fragment of the target protein?

2.2 Have you used any tagging which may interact with the recognition of the epitope?

2.3 How have you selected the sequence of siRNA? Have you tried more than one SiRNA sequence? Could you check if the sequence corresponds correctly toDnmt3L protein?

2.4 Have you checked the KO with Southern blot application?

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Read More

Question

Our customer has responded with the following;   Dear Abcam,   I really can’t waste any more time after something that is not relevant. I can’t waste anymore of my time putting and labeling figures to file a complaint, these figs can’t even make it to the paper, why should I be putting them together. I have run 3 westerns so far and 2 immunos and 1 ChIP, I seriously can’t afford any more time in this. DNMT3L was supposed to be the final bit in the paper and the most important part. The bands are at the perfect size. Gapdh at 37 and DNMT3L at 50 kD. hESCs and hIPSCs have been shown to express it so they are the positive control themselves. All we were looking for was difference in expression between disease and control human ips cells. Since we didn’t find any difference, we looked at localization where we were surprised to find it cytoplasmic. We looked again with different permeabilization protocols, still same results. Your AbReview shows DNMT3L to be nuclear which is correct and that’s how it should be, but its not in our case. So we stained negative control human fibroblast cell lines and again, staining was cytoplasmic where it should have been completely blank. Human fibroblasts can never express DNMT3L. Ofcourse, a secondary only control was used and it was blank, it is an obvious figure to attach, can’t be wasting my time doing it. Again, I can’t be wasting my time putting figures together in a paper like format when the stuff didn’t even work after spending months getting these cells to this stage and running these assays and finding out this. Antibody dilutions for IF have been used at 1:200 which is what is shown in the Abreview by a user for hESCs. Fits perfectly with our stuff. Should not have had a problem. We used BSA and milk both for westerns, BSA was shit and gave tons of background, 10% milk worked great.   I am sorry for the tone but I am extremely disappointed over this product. I have been a faithful abcam user bcoz it has been good with quality for which they demand expensive prices, but DNMT3L has been a bad experience. I can’t waste any more time in something that has not worked and I hope you understand. I demand a full-refund of the antibody purchased and replacement with a new and of course a different DNMT3L antibody that has some chance of being specific.   I wait for your response.   Kind Regards  

Read More
Answer

Thank you for providing some further information. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful. This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab3493. For your information, the internal reference note for this credit is CNXXXX. You can use this credit to refund this troubled customer or to order any of the products which are in the catalogue. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice. I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (lymphoma cell line)
Loading amount
150 µg
Specification
lymphoma cell line
Gel Running Conditions
Reduced Denaturing (10)
Blocking step
Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Feb 24 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (rat lung tissue)
Loading amount
30 µg
Specification
rat lung tissue
Gel Running Conditions
Reduced Denaturing (12% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 14 2009

Application
Western blot
Sample
Armenian Hamster Tissue lysate - whole (hamster muscle tissue)
Loading amount
20 µg
Specification
hamster muscle tissue
Gel Running Conditions
Reduced Denaturing (12% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 28 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Rat Tissue lysate - whole (lung tissue)
Total protein in input
100 µg
Specification
lung tissue
Immuno-precipitation step
Protein A/G

Abcam user community

Verified customer

Submitted Mar 09 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Mouse Tissue lysate - whole (lung tissue)
Total protein in input
100 µg
Specification
lung tissue
Immuno-precipitation step
Protein A/G

Abcam user community

Verified customer

Submitted Mar 09 2009

1-10 of 21 Abreviews or Q&A

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