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: ICC/IF, Flow Cyt, 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), HT-29, and 293T cell lysates. IHC-P: Human colon and spleen tissues. ICC/IF: Wild-type HAP1 cells.
  • General notes

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

     

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

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
ICC/IF 1/250.
Flow Cyt 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. 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.
  • Information by UniProt
  • Database links
  • 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

  • Lanes 1, 5 and 9: Wild-type HAP1 cell lysate (20 µg)
    Lanes 2, 6 and 10: Chk2 knockout HAP1 cell lysate (20 µg)
    Lanes 3, 7 and 11: HeLa cell lysate (20 µg)
    Lanes 4, 8 and 12: HEK293 cell lysate (20 µg)
    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.

  • 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.

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

  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Chk2 with purified ab109413 at 1/230 dilution(10 µg/ml) (red). Cells were fixed with 80% methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 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.

  • Lanes 1: Wild-type HAP1 cell lysate (20 µg)
    Lanes 2: Chk2 knockout HAP1 cell lysate (20 µg)
    Lanes 3: HeLa cell lysate (20 µg)
    Lanes 4: HEK293 cell lysate (20 µg)

    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.

     

  • 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?

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

  • 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

References

This product has been referenced in:
  • Yang F  et al. HPRT1 activity loss is associated with resistance to thiopurine in ALL. Oncotarget 9:2268-2278 (2018). Read more (PubMed: 29416770) »
  • Zhu M  et al. Expression of DNA doublestrand repair proteins in oral leukoplakia and the risk of malignant transformation. Oncol Lett 15:9827-9835 (2018). Read more (PubMed: 29928356) »
See all 8 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted May 13 2013

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

Abcam user community

Verified customer

Submitted May 31 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Jul 09 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted May 02 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton X-100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Feb 19 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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