製品の概要

  • 製品名
    Anti-KDM4C / GASC1 / JMJD2C antibody
    KDM4C / GASC1 / JMJD2C 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to KDM4C / GASC1 / JMJD2C
  • 由来種
    Rabbit
  • アプリケーション
    適用あり: IHC-P, WBmore details
  • 種交差性
    交差種: Mouse, Human
    交差が予測される動物種: Rat, Rabbit, Horse, Guinea pig, Cow, Cat, Dog, Pig
  • 免疫原

    Synthetic peptide corresponding to a region within the internal sequence amino acids 755-804 (CLCNLRGGAL KQTKNNKWAH VMCAVAVPEV RFTNVPERTQ IDVGRIPLQR) of Human KDM4C/ GASC1/ JMJD2C, NP_055876.

  • ポジティブ・コントロール
    • HepG2 cell lysate

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab85454 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 100 kDa (predicted molecular weight: 120 kDa).Can be blocked with Human KDM4C / GASC1 / JMJD2C peptide (ab111662) or KDM4C / GASC1 / JMJD2C peptide (ab155886). Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.

ターゲット情報

  • 機能
    Histone demethylase that specifically demethylates 'Lys-9' and 'Lys-36' residues of histone H3, thereby playing a central role in histone code. Does not demethylate histone H3 'Lys-4', H3 'Lys-27' nor H4 'Lys-20'. Demethylates trimethylated H3 'Lys-9' and H3 'Lys-36' residue, while it has no activity on mono- and dimethylated residues. Demethylation of Lys residue generates formaldehyde and succinate.
  • 組織特異性
    Overexpressed in several esophageal squamous cell carcinomas (ESCs).
  • 配列類似性
    Belongs to the JHDM3 histone demethylase family.
    Contains 1 JmjC domain.
    Contains 1 JmjN domain.
    Contains 2 PHD-type zinc fingers.
    Contains 2 Tudor domains.
  • ドメイン
    The 2 Tudor domains recognize and bind methylated histones. Double Tudor domain has an interdigitated structure and the unusual fold is required for its ability to bind methylated histone tails.
  • 細胞内局在
    Nucleus.
  • Information by UniProt
  • 参照データベース
  • 別名
    • bA146B14.1 antibody
    • GASC 1 protein antibody
    • GASC-1 protein antibody
    • GASC1 antibody
    • Gene amplified in squamous cell carcinoma 1 protein antibody
    • JHDM3C antibody
    • JmjC domain containing histone demethylation protein 3C antibody
    • JmjC domain-containing histone demethylation protein 3C antibody
    • JMJD2C antibody
    • Jumonji domain containing 2C antibody
    • Jumonji domain containing protein 2C antibody
    • Jumonji domain-containing protein 2C antibody
    • Kdm4c antibody
    • KDM4C_HUMAN antibody
    • Lysine (K) specific demethylase 4C antibody
    • Lysine demethylase 4C antibody
    • Lysine-specific demethylase 4C antibody
    • TDRD14C antibody
    • Tudor domain containing 14C antibody
    see all

画像

  • Anti-KDM4C / GASC1 / JMJD2C antibody (ab85454) at 1 µg/ml + HepG2 cell lysate at 10 µg

    Secondary
    HRP conjugated anti-Rabbit IgG at 1/50000 dilution

    Predicted band size: 120 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?



    Gel concentration 6-18%

参考文献

This product has been referenced in:
  • Yu Y  et al. Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer Cell 33:322-336.e8 (2018). Read more (PubMed: 29438700) »
  • Brügger V  et al. Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration. Nat Commun 8:14272 (2017). IF . Read more (PubMed: 28139683) »
See all 5 Publications for this product

レビューと Q&A

1-10 of 16 Abreviews or Q&A

Answer

I have a follow up question based on multiple bands disappearing when the blocking peptide is added. What are the molecular weights of the other bands in your blot? If more than one band disappears in Western blot by peptide/antigen competition, those bands contain the antigenic determinants and could be fragments of the full antigen (protease and phosphatase inhibitors should be included), a complex containing the antigen, or protein isoforms.

I ran a blast on the immunogen sequence given on the datasheet, and KDM4C (alternative name: lysine-specific demethylase 4C) has 4 isoforms that have 100% identity with the immunogen sequence. Links to the information (including varying size) for each isoform are given below:
Isoform 1: http://www.ncbi.nlm.nih.gov/protein/109255247?report=genbank&log$=protalign&blast_rank=15&RID=GS31SNBT013
Isoform 2: http://www.ncbi.nlm.nih.gov/protein/226442889?report=genbank&log$=protalign&blast_rank=26&RID=GS31SNBT013
Isoform 3: http://www.ncbi.nlm.nih.gov/protein/226442893?report=genbank&log$=prottop&blast_rank=3&RID=GS31SNBT013
Isoform 4: http://www.ncbi.nlm.nih.gov/protein/226442897?report=genbank&log$=prottop&blast_rank=2&RID=GS31SNBT013

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Answer

Thank you for your reply.

I have sent you the blocking peptide (https://www.abcam.com/kdm4c--gasc1--jmjd2c-peptide-ab155886.html) free of charge. Your order number is 1236828, and is expected to be delivered Friday, 1/18/2013.

Below is a link to our protocol for the blocking study, which will help determine what bands are specific vs. non-specific:
https://www.abcam.com/index.html?pageconfig=resource&rid=12601

I hope this information is helpful to you. If the 100kDa band remains present when the blocking peptide is used, I will be happy to send you a replacement antibody free of charge.

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Answer


Knockdown at the mRNA level does not necessarily equate to a visible difference at the protein level in the WB band intensity. Since 100kDa is the expected band size for KDM4C, it is very likely that this band is not non-specific but rather a reflection that at the protein level there is not yet a discernible difference in your samples. However, since you suspect that this band is non-specific, I would be happy to send you the blocking peptide for this antibody free of charge to assist in your research and confirm or negate the specificity of the antibody. Additionally, in order to make your western blot more sensitive to changes in protein level, I recommend lowering the primary and secondary antibody concentration and keeping your exposure time to a minimum.

Please let me know if you would be interested in performing the blocking study and I will send you the blocking peptide free of charge. If the 100kDa band remains present when the blocking peptide is used, I will be happy to send you a replacement antibody free of charge. I look forward to your reply.

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Answer

Vielen Dank für Ihre Antwort.

Die Referenznummer Ihrer Gutschrift ist 123456

Bitte setzen Sie sich mit der Apotheke/Reagenzienzentrale in Kontakt, um zu verhindern, dass diese Ihre Gutschrift/die Erstattung ohne Ihr Einverständnisse nutzen

Es tut mir leid, dass der Antikörper nicht so funktioniert hat, wie auf dem Datenblatt angegeben. Ich habe unsere Finanzabteilung beauftragt, eine Gutschrift für Sie auszustellen. Sie können diese Gutschrift auf folgende Weise verwenden:

(1) Gegen die Rechnung dieses Antikörpers verwenden falls diese noch nicht bezahlt wurde
(2) Aufbewahren und gegen eine zukünftige Rechnung verwenden
(3) Wir stellen Ihnen gerne eine komplette Rückerstattung aus, wenn keine offenen Rechnungen mehr ausstehen

Um eine Rückerstattung des Kaufpreises zu erhalten oder um zu bestätigen wie Sie diese Gutschrift benutzen möchten, möchte ich Sie bitten, sich mit unserer Finanzabteilung in Verbindung zu setzen.
Sie können unsere Finanzabteilung per Email unter der Adresse creditcontrol@abcam.com oder telefonisch unter der Nummer 030 896 779 154 erreichen.

Bitte geben Sie die Referenznummer Ihrer Gutschrift immer mit an.

Die Referenznummer ist für Ihre Unterlagen bestimmt. Wir schicken Ihnen die eigentliche Gutschrift per Post oder Email zu. Sie enthält auch die Gutschriftnummer, die mit dem Buchstaben CGB beginnt.

Ich hoffe dass Sie trotz dieser schlechten Erfahrung weiterhin Kunde bei uns bleiben. Bitte zögern Sie nicht sich wieder an uns zu wenden, falls Sie weitere Fragen haben.

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Question
Answer

Leider hat sich mein Verdacht bestätigt: der Antikörper wurde noch nicht in der Maus getestet. ich habe unser Datenblatt dementsprechend korrigiert.

Es ist übrigens sehr interessant, dass der Antikörper nicht kreuzreagiert, da die beiden Proteine in Mensch und Maus zu 100% identisch sind.

Bitte lassen Sie mich wissen ob Sie lieber eine kostenlose Alternative, einen Gutschein oder eine Erstattung des Kaufpreises bevorzugen. Desweiteren müssten ich noch wissen, wann Sie diesen Antikörper gekauft haben.

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Question
Answer


I am very pleased to hear you would like to accept our offer and test these antibodies noted above in IP. This code will give you: 1 free PRIMARY ANTIBODY OR COST OF PRODUCT OFF OF ORDER before the expiration date. To redeem this offer, please submit an Abreview for IP and 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.

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Question

Here are the picture of my WB and the answers to the survey.


1) Abcam product code ab

ab85454 (GR33581-2) and ab56759 (GR60796-1)



3) Description of the problem



ab56759 works in IF but I am unable to make it works in WB.

Ab85454 works neither in IF, nor in WB.

4) Sample preparation: Cumulus cells and embryos, whole cell lysates

Lysis buffer : SDS-PAGE or Laemmli 2X

Protease inhibitors: No

Phosphatase inhibitors No

Reducing agent

Boiling for ≥5 min? Yes, 9 minutes

Protein loaded ug/lane or cells/lane : 35ug

Positive control : HeLa (ab85454), HepG2 (ab56759)

Negative control : Nothing



5) Percentage of gel : I have tried 10%-8%-6%

Type of membrane Nitrocellulose

Protein transfer verified Yes, with rouge de ponceau

Blocking agent and concentration ECL blocking agent 5% or 5% milk in TBST 0.1%

Blocking time 1h30

Blocking temperature RT



6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution : Both tested 1:500

Diluent buffer TBST 0.1% with ECL blocking agent 5% or milk 5%

Incubation time : O/N

Incubation temperature: 4°C



7) Secondary antibody: HRP goat anti-mouse for ab56759 and HRP goat anti-rabbit for ab85454

Concentration or dilution : Very good secondary antibodies, diluted 1:100 000

Diluent buffer TBST 5% milk

Incubation time 1h

Incubation temperature: RT



8) Washing after primary and secondary antibodies:

Buffer

Number of washes 5 washes 5 minutes



9) Detection method Pellicule



10) How many times have you run this staining? 5 times

Do you obtain the same results every time? Yes

What steps have you altered to try and optimize the use of this antibody? Dilution of the antibody. Blocking agent. % of the gel. Transfer time.
Best regards,

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Answer

Thank you for your message and for getting back to me with further details. It is much appreciated.

I am sorry to hear you are disappointed with our products. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

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

1) Tested species:


It would be interesting to know the species of the embryos used in these studies to make sure that the cross-reactivity of the antibody does match with the sample species.

2) Expression levels:

The expression of KDM4C / GASC1 / JMJD2C varies in different section and age of the embryos. You may wish to visit this website for further details:

http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.709425

3) Lysis buffer:

In order to prevent any unwanted degradation of the protein, it is recommended to use a lysis buffer (i.e. RIPA) which contains a mixture of different proteinase inhibitors (i.e.aprotinin, leupeptin, pepstatin A). The recipe for this buffer can be found on our website at:

https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1

4) Blocking:

Try to reduce the blocking time to 1 hr at RT (or overnight at 4oC) since too stringent/long blocking can reduce/eliminate the signal.

I hope this will be useful for you. Should you still have any problem after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful fromab85454 and ab71732. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I apologize for the inconvenience and as requestedam pleased to provide the alternativefree of charge replacements in compensation. I have issued the following free of chargeorder:

Order number:1088302
1 X vial ab123900
1 X vial ab4812

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

Please note that the free of charge replacement vials are 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.

Regarding PKN1 antibody ab47535, I am pleased this is now working for you. I understand your concerns, it is regrettable thatthere were some initial difficulties with this. Please note that any concentrations of dilutions stated on the datasheet are a guideline only, and this will sometimes require further optimization for individual experiments and sample types.

I wish you the best of luck with your research. If you require anything further, please do not hesitate to contact us.

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Answer

Thank you for your response.

I have checked our system which stores all the incoming and outgoing e-mails and I can see that in the initial correspondence, 3 products (ab47535, ab71732 and ab85454) were reported to be faulty.

Could you please update Kate or myself how the other antibodies perform after following some recommendations/tips made by my colleague?

Would you like to get alterative products for ab85454 and ab71732 - but not ab47535?

I look forward to hearing from you and solve this issue as soon as possible.

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1-10 of 16 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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