Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-ALKBH5 antibody [EPR18958] (ab195377)

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Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH5 antibody [EPR18958] (ab195377)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH5 antibody [EPR18958] (ab195377)
  • Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
  • Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
  • Anti-ALKBH5 antibody [EPR18958] (ab195377)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR18958] to ALKBH5
  • Suitable for: WB, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-ALKBH5 antibody [EPR18958]
    See all ALKBH5 primary antibodies

  • Description

    Rabbit monoclonal [EPR18958] to ALKBH5

  • Host species

    Rabbit

  • Specificity

    ab195377 has been tested in IHC-P on Human, Mouse and Rat testis sections. It worked well on Human, but not on the Mouse or Rat sections, under our experimental conditions.

  • Tested applications

    Suitable for: WB, IHC-Pmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Predicted to work with: Sheep, Goat, Horse, Guinea pig, Cow, Cat, Dog, Pig, Non human primates

  • Immunogen

    Recombinant fragment within Human ALKBH5 aa 50-350. The exact sequence is proprietary.
    Database link: Q6P6C2

  • Positive control

    • WB: Wild type MEF, HeLa, HepG2 , PC-12, NIH/3T3, Jurkat, Neuro-2a, HEK-293T and F9 whole cell lysates. IHC-P: Human testis and kidney tissues.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

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

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR18958

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab195377 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
WB 1/1000. Detects a band of approximately 35-44 kDa (predicted molecular weight: 44 kDa).
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Suitable for human tissues only.

Target

  • Relevance

    Dioxygenase that demethylates RNA by oxidative demethylation: specifically demethylates N(6)-methyladenosine (m6A) RNA, the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Requires molecular oxygen, alpha-ketoglutarate and iron. Demethylation of m6A mRNA affects mRNA processing and export and is required for spermatogenesis.

  • Cellular localization

    Nucleus speckle

  • Database links

    see all

  • Alternative names

    • ABH5 antibody
    • AlkB, alkylation repair homolog 5 (E. coli) antibody
    • AlkB, alkylation repair homolog 5 antibody
    • Alkylated DNA repair protein alkB homolog 5 antibody
    • Alpha ketoglutarate dependent dioxygenase alkB homolog 5 antibody
    • OFOXD antibody
    • OFOXD1 antibody
    • Oxoglutarate and iron-dependent oxygenase domain containing antibody
    • Probable alpha ketoglutarate dependent dioxygenase ABH5 antibody
    • RNA demethylase ALKBH5 antibody
    see all

Images

  • Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    All lanes : Anti-ALKBH5 antibody [EPR18958] (ab195377) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : ALKBH5 knockout HEK-293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 44 kDa
    Observed band size: 48 kDa
    why is the actual band size different from the predicted?



    Lanes 1 - 2: Merged signal (red and green). Green - ab195377 observed at 48 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab195377 was shown to react with ALKBH5 in wild-type HEK-293T cells in western blot with loss of signal observed in ALKBH5 knockout cell line ab266762 (ALKBH5 knockout cell lysate ab257349). Wild-type and ALKBH5 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab195377 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH5 antibody [EPR18958] (ab195377)

    Immunohistochemical analysis of paraffin-embedded human testis tissue labeling ALKBH5 with ab195377 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining in human testis (PMID: 21264265; PMID: 27590511; PMID: 17979886) is observed. Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALKBH5 antibody [EPR18958] (ab195377)

    Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling ALKBH5 with ab195377 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining in human kidney (PMID: 21264265) is observed. Counter stained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Lanes 1-2 : Anti-ALKBH5 antibody [EPR18958] (ab195377) at 1/1000 dilution
    Lanes 3-5 : Anti-ALKBH5 antibody [EPR18958] (ab195377) at 1/2000 dilution

    Lane 1 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
    Lane 2 : NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate
    Lane 3 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
    Lane 4 : Neuro-2a (mouse neuroblastoma cell line) whole cell lysate
    Lane 5 : F9 (mouse embryonic testicular cancer cell line) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 44 kDa
    Observed band size: 35-44 kDa why is the actual band size different from the predicted?



    Exposure time : Lanes 1 and 2: 15 seconds; Lanes 3 and 4: 26 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The molecular mass observed is consistent with the literature (PMID 21264265).

  • Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Western blot - Anti-ALKBH5 antibody [EPR18958] (ab195377)
    All lanes : Anti-ALKBH5 antibody [EPR18958] (ab195377) at 1/5000 dilution

    Lane 1 : Wild type MEF whole cell lysate
    Lane 2 : ALKBH5 knockout MEF whole cell lysate
    Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 44 kDa
    Observed band size: 35-44 kDa why is the actual band size different from the predicted?


    Exposure time: 70 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The Wt and ALKBH5 KO cell lysates were kindly provided by an anonymous collaborator.

    The molecular mass observed is consistent with the literature (PMID 21264265).

  • Anti-ALKBH5 antibody [EPR18958] (ab195377)
    Anti-ALKBH5 antibody [EPR18958] (ab195377)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (14)

Publishing research using ab195377? Please let us know so that we can cite the reference in this datasheet.

ab195377 has been referenced in 14 publications.

  • Zhu B  et al. Total Panax notoginseng saponin inhibits vascular smooth muscle cell proliferation and migration and intimal hyperplasia by regulating WTAP/p16 signals via m6A modulation. Biomed Pharmacother 124:109935 (2020). WB ; Human . PubMed: 31986407
  • Li G  et al. Bone-derived mesenchymal stem cells alleviate compression-induced apoptosis of nucleus pulposus cells by N6 methyladenosine of autophagy. Cell Death Dis 11:103 (2020). WB ; Human . PubMed: 32029706
  • Jabs S  et al. Impact of the gut microbiota on the m6A epitranscriptome of mouse cecum and liver. Nat Commun 11:1344 (2020). WB ; Mouse . PubMed: 32165618
  • Cen S  et al. TRAF4 acts as a fate checkpoint to regulate the adipogenic differentiation of MSCs by activating PKM2. EBioMedicine 54:102722 (2020). WB ; Human . PubMed: 32268273
  • Shen C  et al. RNA Demethylase ALKBH5 Selectively Promotes Tumorigenesis and Cancer Stem Cell Self-Renewal in Acute Myeloid Leukemia. Cell Stem Cell N/A:N/A (2020). WB ; Human . PubMed: 32402250
View all Publications for this product

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Western blot abreview for Anti-ALKBH5 antibody [EPR18958]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Pig Cell lysate - whole cell (Swine Testicle cells (ST))
Gel Running Conditions
Reduced Denaturing (10% polyacrylamide gel)
Loading amount
15 µg
Specification
Swine Testicle cells (ST)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Oct 04 2018

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