Anti-NF-kB p65 (acetyl K310)抗体- ChIP Grade (ab19870)

概述

  • 产品名称
    Anti-NF-kB p65 (acetyl K310)抗体- ChIP Grade
    参阅全部 NF-kB p65 一抗
  • 描述
    兔多克隆抗体to NF-kB p65 (acetyl K310) - ChIP Grade
  • 宿主
    Rabbit
  • 经测试应用
    适用于: WB, IP, Dot blot, ICC, ChIPmore details
  • 种属反应性
    与反应: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide corresponding to Human NF-kB p65 aa 300-400 (internal sequence) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab20612)

  • 阳性对照
    • This antibody gave a positive signal in Rat lung tissue lysate.

应用

Our Abpromise guarantee covers the use of ab19870 in the following tested applications.

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

应用 Ab评论 说明
WB Use a concentration of 2.5 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa). Collaborator data suggests that immunoprecipitation of this antibody prior to Western blotting is required to obtain the best results (see images)
IP Use a concentration of 2.5 µg/ml.
Dot blot Use at an assay dependent dilution.
ICC Use at an assay dependent dilution. In ICC/IF ab19870 recognizes various acetylated nuclear protein(s), as the signal is also observed in control cells; the signal in ICC is HDACi-dependent.
ChIP Use at an assay dependent concentration. PubMed: 22249179

靶标

  • 功能
    NF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1.
  • 序列相似性
    Contains 1 RHD (Rel-like) domain.
  • 结构域
    the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
  • 翻译后修饰
    Ubiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response.
    Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes.
    Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity.
    Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at Lys-122 enhances DNA binding and impairs association with NFKBIA. Acetylation at Lys-310 is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of 'Lys-310'.
  • 细胞定位
    Nucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction.
  • Information by UniProt
  • 数据库链接
  • 别名
    • Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
    • MGC131774 antibody
    • NF kappa B p65delta3 antibody
    • NFKB3 antibody
    • Nuclear Factor NF Kappa B p65 Subunit antibody
    • Nuclear factor NF-kappa-B p65 subunit antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 antibody
    • OTTHUMP00000233473 antibody
    • OTTHUMP00000233474 antibody
    • OTTHUMP00000233475 antibody
    • OTTHUMP00000233476 antibody
    • OTTHUMP00000233900 antibody
    • p65 antibody
    • p65 NF kappaB antibody
    • p65 NFkB antibody
    • relA antibody
    • TF65_HUMAN antibody
    • Transcription factor p65 antibody
    • v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65)) antibody
    • V rel avian reticuloendotheliosis viral oncogene homolog A antibody
    • v rel reticuloendotheliosis viral oncogene homolog A (avian) antibody
    • V rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B cells 3, p65 antibody
    see all

图片

  • Lanes 1-2 : Coomassie stain
    Lanes 3-4 : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1/500 dilution

    Lanes 1 & 3 : Acetylated p65 protein
    Lanes 2 & 4 : K310 mutant protein

    Lysates/proteins at 2 µg per lane.

    Secondary
    Lanes 3-4 : IRDye® 800CW Goat anti-Rabbit IgG (H + L) at 1/15000 dilution

    Predicted band size: 65 kDa



    Observed band size: 75kDa

    The p65 band runs higher in this blot because the protein contains a myc-tag.

  • All lanes : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1/1000 dilution

    Lane 1 : HCT116 whole cell lysate treated with DMSO for 24 hrs (control)
    Lane 2 : HCT116 whole cell lysate treated with 2 µM SAHA for 24 hrs
    Lane 3 : MEF whole cell lysate treated with DMSO for 24 hrs (control)
    Lane 4 : MEF whole cell lysate treated with 2 µM SAHA for 24 hrs

    Lysates/proteins at 60 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 65 kDa
    Observed band size: 65 kDa
    Additional bands at: 140 kDa (possible non-specific binding), 15 kDa (possible non-specific binding), 40 kDa (possible non-specific binding), 45 kDa (possible non-specific binding), 90 kDa (possible non-specific binding)


    Exposure time: 2 minutes

    See Abreview

  • Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3h at room temperature. Exposure time: 75 min normal ECL. This Dot blot demonstrates that ab19870 recognized upto 10ng of purified peptide on a PVDF membrane.

  • Western Blot with ab19870 after p65 Immunoprecipitation: rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3 hours at room temperature. Exposure time: 1 min normal ECL. Tested samples: nuclear extracts (180 µg) of immortalized p65-/- mouse cells, complemented with the empty vector (pRRL), wild-type p65 (Wt) and non-acetylatable K310 (K310R). The samples tested were treated with deacetylase inhibitors HDACi (TSA + Nicotinamide) and TNF-alpha. The samples were immunoprecipitated with 2µg of alpha-p65 and subsequently analysed by Western blot with Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870). Predicted band size = 65kDa, Observed band size = 75kDa. The p65 band runs higher in this SDS-PAGE blot as it contains a myc-tag. 

  • All lanes : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 2.5 µg/ml

    Lane 1 : pRRL untreated
    Lane 2 : pRRL HDACi
    Lane 3 : pRRL HDACi + TNF
    Lane 4 : Wt untreated
    Lane 5 : Wt HDACi
    Lane 6 : Wt HDACi + phorbol myristate acetate
    Lane 7 : K310R untreated
    Lane 8 : K310R HDACi
    Lane 9 : K310R HDACi + phorbol myristate acetate

    Lysates/proteins at 75 µg per lane.

    Developed using the ECL technique.

    Predicted band size: 65 kDa
    Observed band size: 75 kDa
    why is the actual band size different from the predicted?


    Exposure time: 1 hour


    ab19870 recognizes Rabbit polyclonal to NF-kB p65 (acetyl K310) specifically at ~75kDa (indicated by the arrow) is this SDS-PAGE blot. The p65 band runs higher than 65kDa in this SDS-PAGE blot as it contains a myc-tag. We are sure that the band at ~75kDa is p65 since p65 specific antibodies detect the same band in IP and WB and there is no signal in the p65 knock-out cell line with ab19870. A number of additonal bands are recognized by ab19870 when tested with endogenous p65 from whole cell extracts, we do not know the identity of these bands.

    Tested samples: nuclear extracts (75µg) of immortalized p65-/- mouse cells, complemented with the empty ve

  • Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1 µg/ml + Lung (Rat) Tissue Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 65 kDa
    Observed band size: 72 kDa why is the actual band size different from the predicted?
    Additional bands at: 15 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 4 minutes

文献

This product has been referenced in:
  • Fan S  et al. JNK and NF-?B signaling pathways are involved in cytokine changes in patients with congenital heart disease prior to and after transcatheter closure. Exp Ther Med 15:1525-1531 (2018). WB ; Human . Read more (PubMed: 29434738) »
  • Zhang Z  et al. Mouse macrophage specific knockout of SIRT1 influences macrophage polarization and promotes angiotensin II-induced abdominal aortic aneurysm formation. J Genet Genomics 45:25-32 (2018). WB ; Mouse . Read more (PubMed: 29396144) »
See all 42 Publications for this product

客户评价及客户问答

1-4 of 4 Q&A

Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

I have read the details you have kindly provided and have following further questions for better understanding of the problem;

- Could you fill in the attached questionnaire? Please provide the IP details also.

Thank you very much for your cooperation. I will look forward to hearing from you soon.

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Answer

Thank you for your reply.

I have arranged a free of charge vial of ab52175 to ship out to Cedarlane on their next order from us as a replacement for the faulty ab19870 that you ordered through them on reference number is 389794. When they get the new antibody they will send it to you, so please confirm your shipping address with Cedarlane.

I hope this information helps. Please contact us with any other questions.

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Answer

Thank you for your reply and for clarifying those additional protocol details.

I may be able to look up your order details for you if you can provide more information. What institution are you working in? Did you order through a distributor?

The protocol seems fine and as long as the antibody was purchased within the past 6 months I'd be happy to offer you a replacement or refund. I know that you said you'd be interested in trying ab52175, so please let me know your original order details so that I could work on getting that to you.

I hope this information helps. Please contact us with any other questions.

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Answer

Thank you very much for your interest in ab19870.

To our knowledge,ab19870 has not been tested in IHC-P. Therefore, I can offer a discount off a future purchase if you buyab19870 now, test it inIHC-P and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free PRIMARY ANTIBODY.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and testab19870 in IHC-P. I will then send a discount code. This code must be issued before purchasingab19870 so please wait for my reply before ordering.

2. Purchaseab19870 either by phone, fax, or online (www.abcam.com).

3. Test it in IHC-P.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any PRIMARY ANTIBODY ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even ifab19870 turns out to be unsuitable for IHC-P, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

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

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