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  1. Link

    chk2-antibody-epr4325-ab109413.pdf

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Epigenetics and Nuclear Signaling DNA / RNA DNA Damage & Repair DNA Damage Response Chk1 / Chk2
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Chk2 antibody [EPR4325] (ab109413)

  • Datasheet
Reviews (9)Q&A (1)References (17)

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Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Immunocytochemistry - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Flow Cytometry (Intracellular) - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Immunoprecipitation - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Immunocytochemistry - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR4325] (ab109413)
  • Anti-Chk2 antibody [EPR4325] (ab109413)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR4325] to Chk2
  • Suitable for: Flow Cyt (Intra), WB, IP, IHC-P, ICC
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-Chk2 antibody [EPR4325]
    See all Chk2 primary antibodies
  • Description

    Rabbit monoclonal [EPR4325] to Chk2
  • Host species

    Rabbit
  • Tested applications

    Suitable for: Flow Cyt (Intra), WB, IP, IHC-P, ICCmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment within Human Chk2 aa 1-200. The exact sequence is proprietary.
    Database link: O96017

  • Positive control

    • WB: HeLa (untreated and treated with gamma irradiation), HAP1, CHEK2, HEK293, MDA-MB-231, HT-29, and 293T cell lysates. IHC-P: Human colon and spleen tissues. ICC/IF: Wild-type HAP1 cells. Flow Cyt (intra): HeLa IP: HeLa whole cell lysate.
  • 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.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

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. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.05% Sodium azide
    Constituents: 0.1% BSA, 40% Glycerol (glycerin, glycerine), 9.85% Tris glycine, 50% Tissue culture supernatant
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR4325
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • DNA Damage Response
    • Chk1 / Chk2
    • Cancer
    • Cell cycle
    • Kinases/phosphatases
    • Other

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-Chk2 antibody [EPR4325] (ab200762)
    • Anti-Chk2 antibody [EPR4325] - BSA and Azide free (ab227998)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human CHEK2 (Chk2) knockout HeLa cell line (ab264815)
  • KO cell lysates

    • Human CHEK2 (Chk2) knockout HeLa cell lysate (ab257104)
  • Positive Controls

    • HT-29 whole cell lysate (ab3952)
  • Recombinant Protein

    • Recombinant human Chk2 protein (Active) (ab62285)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab109413 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
Flow Cyt (Intra)
Use at an assay dependent concentration.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.  

WB (5)
1/50000 - 1/200000. Detects a band of approximately 62 kDa (predicted molecular weight: 61 kDa).
IP
1/10 - 1/100.
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

antigen retrieval is recommended.

ICC
1/100 - 1/250.
Notes
Flow Cyt (Intra)
Use at an assay dependent concentration.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.  

WB
1/50000 - 1/200000. Detects a band of approximately 62 kDa (predicted molecular weight: 61 kDa).
IP
1/10 - 1/100.
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

antigen retrieval is recommended.

ICC
1/100 - 1/250.

Target

  • Function

    Regulates cell cycle checkpoints and apoptosis in response to DNA damage, particularly to DNA double-strand breaks. Inhibits CDC25C phosphatase by phosphorylation on 'Ser-216', preventing the entry into mitosis. May also play a role in meiosis. Regulates the TP53 tumor suppressor through phosphorylation at 'Thr-18' and 'Ser-20'.
  • Tissue specificity

    High expression is found in testis, spleen, colon and peripheral blood leukocytes. Low expression is found in other tissues.
  • Involvement in disease

    Defects in CHEK2 are associated with Li-Fraumeni syndrome 2 (LFS2) [MIM:609265]; a highly penetrant familial cancer phenotype usually associated with inherited mutations in p53/TP53.
    Defects in CHEK2 may be a cause of susceptibility to prostate cancer (PC) [MIM:176807]. It is a malignancy originating in tissues of the prostate. Most prostate cancers are adenocarcinomas that develop in the acini of the prostatic ducts. Other rare histopathologic types of prostate cancer that occur in approximately 5% of patients include small cell carcinoma, mucinous carcinoma, prostatic ductal carcinoma, transitional cell carcinoma, squamous cell carcinoma, basal cell carcinoma, adenoid cystic carcinoma (basaloid), signet-ring cell carcinoma and neuroendocrine carcinoma.
    Defects in CHEK2 are found in some patients with osteogenic sarcoma (OSRC) [MIM:259500].
  • Sequence similarities

    Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. CHK2 subfamily.
    Contains 1 FHA domain.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications

    Phosphorylated by PLK4.
  • Cellular localization

    Nucleus; Nucleus. Isoform 10 is present throughout the cell and Nucleus > PML body. Nucleus > nucleoplasm. Recruited into PML bodies together with TP53.
  • Target information above from: UniProt accession O96017 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 11200 Human
    • Omim: 604373 Human
    • SwissProt: O96017 Human
    • Unigene: 291363 Human
    • Unigene: 505297 Human
    • Alternative names

      • CDS 1 antibody
      • Cds1 antibody
      • Cds1 homolog antibody
      • Checkpoint kinase 2 antibody
      • Checkpoint like protein CHK2 antibody
      • CHEK 2 antibody
      • Chek2 antibody
      • Chk 2 antibody
      • CHK2 checkpoint homolog (S. pombe) antibody
      • CHK2 checkpoint homolog antibody
      • CHK2_HUMAN antibody
      • hCds1 antibody
      • HuCds 1 antibody
      • LFS 2 antibody
      • LFS2 antibody
      • PP1425 antibody
      • RAD 53 antibody
      • RAD53 antibody
      • Rad53 homolog antibody
      • Serine/threonine protein kinase Chk2 antibody
      • Serine/threonine-protein kinase Chk2 antibody
      see all

    Images

    • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      All lanes : Anti-Chk2 antibody [EPR4325] (ab109413) at 1/1000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : CHEK2 knockout HeLa cell lysate
      Lane 3 : HEK-293 cell lysate
      Lane 4 : MDA-MB-231 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 61 kDa
      Observed band size: 68 kDa why is the actual band size different from the predicted?



      Lanes 1-4: Merged signal (red and green). Green - ab109413 observed at 68 kDa. Red - loading control ab8245 observed at 37 kDa.

       ab109413 Anti-Chk2 antibody [EPR4325] was shown to specifically react with Chk2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264815 (knockout cell lysate ab257104) was used. Wild-type and Chk2 knockout samples were subjected to SDS-PAGE. ab109413 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

       

    • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      Lanes 1-4 : Anti-Chk2 antibody [EPR4325] (ab109413) at 1/50000 dilution
      Lanes 5-8 : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution

      Lanes 1 & 5 : Wild-type HAP1 cell lysate
      Lanes 2 & 6 : Chk2 knockout HAP1 cell lysate
      Lanes 3 & 7 : HeLa cell lysate
      Lanes 4 & 8 : HEK293 cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 61 kDa



      Lanes 1, 2, 3 and 4: Green signal from target -  ab109413 observed at 62 kDa
      Lanes 5, 6, 7 and 8: Red signal from loading control - ab8245 observed at 37 kDa
      Lanes 9, 10, 11 and 12: Merged (red and green) signal

      ab109413 was shown to specifically react with Chk2 when Chk2 knockout samples were used. Wild-type and Chk2 knockout samples were subjected to SDS-PAGE. ab109413 and ab8245 (loading control to GAPDH) were diluted 1/50 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

    • Immunocytochemistry - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunocytochemistry - Anti-Chk2 antibody [EPR4325] (ab109413)

      ab109413 staining Chk2 in wild-type HAP1 cells (top panel) and Chk2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5 minutes), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab109413 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (ab109413)

      Immunohistochemical analysis of paraffin-embedded human colon tissue using ab109413 at a 1/100 dilution.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Flow Cytometry (Intracellular) - Anti-Chk2 antibody [EPR4325] (ab109413)
      Flow Cytometry (Intracellular) - Anti-Chk2 antibody [EPR4325] (ab109413)

      Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Chk2 with purified ab109413 at 1/230 dilution (10µg/ml) (red). Cells were fixed with 80% methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluorr® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

       

       

    • Immunoprecipitation - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunoprecipitation - Anti-Chk2 antibody [EPR4325] (ab109413)

      Purified ab109413 at 1/50 dilution (2µg) immunoprecipitating Chk2 in HeLa whole cell lysate.
      Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
      Lane 2 (+): ab109413 + HeLa whole cell lysate.
      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109413 in HeLa whole cell lysate.
      VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
      Blocking Buffer and concentration: 5% NFDM/TBST.
      Diluting buffer and concentration: 5% NFDM/TBST.
      Observed band size: 62 kDa

    • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      All lanes : Anti-Chk2 antibody [EPR4325] (ab109413)

      Lane 1 : Wild-type HAP1 cell lysate
      Lane 2 : Chk2 knockout HAP1 cell lysate
      Lane 3 : HeLa cell lysate
      Lane 4 : HEK293 cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 61 kDa



      Lanes 1 - 4: Merged signal (red and green). Green - ab109413 observed at 64 kDa. Red - loading control, ab8245, observed at 37 kDa.

      This western blot image is a comparison between ab109413 and a competitor's rabbit polyclonal antibody.

       

    • Immunocytochemistry - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunocytochemistry - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunocytochemistry analysis of HT-29 (human colorectal adenocarcinoma epithelial cell) labeling Chk2 with purified ab109413 at 1/500 dilution. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. was used as counterstain. Nuclei were stained blue with DAPI.
      Negative control: PBS instead of the primary antibody.
    • Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      Western blot - Anti-Chk2 antibody [EPR4325] (ab109413)
      All lanes : Anti-Chk2 antibody [EPR4325] (ab109413) at 1/50000 dilution

      Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with gamma irradiation
      Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
      Lane 3 : HT-29 (human colorectal adenocarcinoma cell line) cell lysate
      Lane 4 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 61 kDa
      Observed band size: 62 kDa why is the actual band size different from the predicted?

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Chk2 antibody [EPR4325] (ab109413)

      Immunohistochemical analysis of paraffin-embedded human spleen tissue using ab109413 at a 1/100 dilution.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR4325] (ab109413)
      Immunocytochemistry/ Immunofluorescence - Anti-Chk2 antibody [EPR4325] (ab109413)Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

      ab109413 (1/500) staining Chk2 in HeLa (human epithelial cell line from cervix adenocarcinoma) cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see Abreview.

      See Abreview

    • Anti-Chk2 antibody [EPR4325] (ab109413)
      Anti-Chk2 antibody [EPR4325] (ab109413)

    Protocols

    • Flow cytometry protocols
    • Immunoprecipitation protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet download

      Download

    References (17)

    Publishing research using ab109413? Please let us know so that we can cite the reference in this datasheet.

    ab109413 has been referenced in 17 publications.

    • Li Y  et al. ITGB1 enhances the Radioresistance of human Non-small Cell Lung Cancer Cells by modulating the DNA damage response and YAP1-induced Epithelial-mesenchymal Transition. Int J Biol Sci 17:635-650 (2021). PubMed: 33613118
    • Ngo GHP  et al. UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair. Nat Commun 12:3849 (2021). PubMed: 34158508
    • Jiang Y  et al. LINC01094 triggers radio-resistance in clear cell renal cell carcinoma via miR-577/CHEK2/FOXM1 axis. Cancer Cell Int 20:274 (2020). PubMed: 32595418
    • Zhao Y  et al. A Germline CHEK2 Mutation in a Family with Papillary Thyroid Cancer. Thyroid 30:924-930 (2020). PubMed: 32041497
    • Sun L  et al. Role of DNA damage in the progress of chronic tubule-interstitial injury. Mol Med Rep 22:1081-1089 (2020). PubMed: 32626982
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-10 of 10 Abreviews or Q&A

    Western blot abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa cell lysate)
    Loading amount
    22 µg
    Specification
    HeLa cell lysate
    Treatment
    +/- 10 Gy IR
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 13 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Mouse vascular smooth muscle cells)
    Permeabilization
    Yes - NP40
    Specification
    Mouse vascular smooth muscle cells
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 07 2022

    Western blot abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - nuclear (Mouse vascular smooth muscle cells)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    30 µg
    Treatment
    10 uM Etoposide for 12 hours
    Specification
    Mouse vascular smooth muscle cells
    Blocking step
    Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 07 2022

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (U2OS)
    Permeabilization
    Yes - NP40
    Specification
    U2OS
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 07 2022

    Western blot abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Hela and Hela Chk2 knockout cell lines)
    Gel Running Conditions
    Reduced Non-Denaturing (Native) (4-12% BIS-TRIS gradient, MOPS running buffer)
    Loading amount
    20 µg
    Specification
    Hela and Hela Chk2 knockout cell lines
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Nadeem Shaikh

    Verified customer

    Submitted May 11 2021

    Western blot abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - nuclear (Vascular smooth muscle cell)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    25 µg
    Treatment
    100 uM H2O2 2 hours
    Specification
    Vascular smooth muscle cell
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 31 2017

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    5% BSA + 1% NDS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Permeabilization
    Yes - 0.1% Triton X-100
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 09 2013

    Western blot abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Hela, HT29)
    Loading amount
    30 µg
    Specification
    Hela, HT29
    Treatment
    see legend
    Gel Running Conditions
    Reduced Denaturing (7% Tris acetate)
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 02 2013

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Chk2 antibody [EPR4325]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% Triton X-100 in PBS
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    DR. Kirk Mcmanus

    Verified customer

    Submitted Feb 19 2013

    Question


    Can you please provide with the exact reaction conditions for western blots with the antibody
    ab109413 (Anti-Chk2).
    I did not get any results that resembled the graph that you have on the website and was wondering if it was the conditions.

    Read More

    Abcam community

    Verified customer

    Asked on Feb 06 2013

    Answer

    Thank you for contacting us.

    Below you can find the tested WB protocol.

    Protocols: Western Blot

    Cell Lysis and Western Blotting Protocol


    http://www.epitomics.com/pdf/westerndata.pdf

    1. Solutions and Reagents
    1.1. 10X Cell Lysis Buffer (Cat #: http://www.epitomics.com/products/product_info/3500-1)

    - Add Phenylmethylsulfonyl fluoride (PMSF) toCEll Lysis Buffer to a final concentration of 1mM. (Stock solution 100mM PMSF) NOTE : Add fresh before each use

    - Add protease and phosphatase inhibitors.

    - 1X protease inhibitor mixture consists of 2 mM AEBSF, 1 mM EDTA, 130 μM bestatin, 14 μM E-64, and 0.3 μM aprotinin.

    1.2. 2X Laemmli Sample Buffer: 62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% Bromophenol Blue, 710 mM beta-mercaptoethanol

    1.3. TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) TBST is Tris Base Saline buffer with 0.1% Tween-20

    1.4. 5% NFDM: Use 25 g non-fat dried milk in 500 ml TBST

    1.5. 5% bovine serum albumin (BSA): Use 25 g in 500 ml TBST

    1.6. Goat anti-rabbit HRP antibody

    1.7. Chemiluminesence reagents; such as ECL materials and film or imaging system for detection

    2. Cell Lysis and Western Blotting Protocol
    Westerns are performed using cell lysates from harvested cells.

    2.1. Adherent cells

    2.1.1 Grow cells to ˜90% confluency.
    2.1.2 Wash cells twice with TBS to remove media
    2.1.3 Add the appropriate volume of 1x Cell lysis buffer (ex: 3ml to a T175 flask)
    2.1.4 Transfer cell lysates to Eppendorf tubes and sonicate for 10-15 seconds.
    2.1.5 Spin at 14,000 rpm, 4 °C for 10 minutes.

    2.2. Suspension cells

    2.2.1 Pipette cells gently into a conical tube and centrifuge 10 min. at 1000 rpm
    2.2.2 Wash cells twice with TBS
    2.2.3 Add the appropriate volume of 1x Cell lysis buffer and transfer to Eppendorf tubes (ex: 1ml for 1X10 7 cells)
    2.2.4 Sonicate for 10-15 seconds.
    2.2.5 Spin at 14,000 rpm, 4 °C for 10 minutes.

    2.3. Remove a small volume (50 ul) to perform a protein assay. Determine the protein concentration for each cell lysate.

    2.4. To the remaining volume of cell lysate, add an equal volume of 2X Laemmli Sample Buffer (final concentration of 1 mg/ml).

    2.5. Boil each cell lysate in sample buffer at 100 °C for 5 min and aliquot. Use a 26-gauge needle to shear released chromatin. Store lysates at -20 °C. Note: Aliquot cell lysates (50-100 μl) in order to avoid repeat freeze/thaw cycles.

    2.6. Defrost tubes containing cell lysate at 37 °C. Centrifuge at 14,000 rpm in a microcentrifuge for 5 min.

    2.7. Load equal amounts (10-20 μg) cell lysate onto SDS-PAGE gels using gel loading tips, along with molecular weight markers.

    2.8. Run gels and transfer for western blotting.

    3. Western Blotting

    3.1. Block nitrocellulose for 1 hour at room temperature or overnight at 4°C using 5% BSA or 5% NFDM.

    3.2.Incubate nitrocellulose with appropriate dilutions of primary antibody in 5% BSA or 5% NFDM overnight at 4°C or for 2 hours at room temperature

    3.3. Wash nitrocellulose with three 5-min washes using TBST.

    3.4.Incubate nitrocellulose with goat anti-rabbit HRP antibody, diluted to 1:500 to 100,000 in 5% BSA or 5% NFDM, for 1-2 hours at room temperature.

    3.5. Wash nitrocellulose in 3 washes of TBST, then rinse in TBS prior to addition of chemiluminesence reagents.

    3.6. Remove excess chemiluminescence reagent and sandwich nitrocellulose blot in any type of transparent plastic wrap (plastic cling wrap, transparent sheet protector, etc.).

    3.7. Acquire image using darkroom development techniques.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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    Abcam Scientific Support

    Answered on Feb 06 2013

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