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Mre11 Antibody

Catalog # NB100-142 | Novus Biologicals a Bio-Techne Brand
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NB100-142
NB100-142SS

Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Human, Mouse, Rat, Chicken, Hamster

Applications

Chromatin Immunoprecipitation (ChIP), ELISA, Flow Cytometry, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockdown Validated, Knockout Validated, Proximity Ligation Assay, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Concentration

This product is unpurified. The exact concentration of antibody is not quantifiable.

Product Summary for Mre11 Antibody

Immunogen

Mre11 Antibody is made to a full length human Mre11 protein. [Uniprot: P49959]

Predicted Species

Bat (92%), Bovine (92%), Canine (94%), Chimpanzee (100%), Equine (92%), Feline (94%), Primate (100%). Backed by our 100% Guarantee.

Reactivity Notes

Predicted cross-reactivity based on sequence identity: Gorilla (100%), Chimpanzee (100%), Gibbon (99%), Marmoset (96%), Canine (94%), Feline (94%), Panda (94%), Equine (92%), Bovine (92%), Bat (92%).

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

81 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Mre11 Antibody

Simple Western: Mre11 Antibody [NB100-142] - Image shows a specific band for Mre11 in 0.5 mg/mL of HeLa lysate. A band appears at the theoretical molecular weight of 81 kDa for this product. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Immunocytochemistry/Immunofluorescence: Mre11 Antibody [NB100-142] - IF analysis of Mre11 on HeLa. Image from verified customer review.
Knockdown Validated: Mre11 Antibody [NB100-142] - Mre11 and NBS1 contribute to OriP replication and EBV episome stability. D98/HR1 cells were transfected with siRNA for Mre11 or NBS1, or Luciferase control and assayed by immunoblot (IB) with antibodies specific for Mre11 (left pane) or NBS1 (right panel), or PCNA (lower panel) as a loading control. Image collected and cropped by CiteAb from the following publication (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001257) licensed under a CC-BY license.

Applications for Mre11 Antibody

Application
Recommended Usage

ELISA

reported in scientific literature (PMID 16788144)

Immunoblotting

reported in scientific literature (PMID 28115467)

Immunocytochemistry/ Immunofluorescence

1:200. Use reported in scientific literature (PMID 26774475)

Immunohistochemistry

1:10 - 1:500

Immunohistochemistry-Frozen

reported in scientific literature (PMID 24349281)

Immunohistochemistry-Paraffin

1:10 - 1:500. Use reported in scientific literature (PMID 21279473)

Immunoprecipitation

3 uL

Knockout Validated

reported in scientific literature (VanCevska et al)

Proximity Ligation Assay

reported in scientific literature (PMID 32780723)

Simple Western

1:250

Western Blot

1:5000
Application Notes
In Western blot, a band can be seen at ~ 81 kDa. For ICC/IF, this antibody has been used with methanol-fixed IMR90 primary human fibroblasts. For IP, the suggested working dilution is 3 ul for immunoprecipitation of 3X10^6 cells. Co-IP application has been reported in scientific literature (PMID: 22190719) In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Separated by Size-Wes, Sally Sue/Peggy Sue.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 4 reviews rated 4.5 using NB100-142 in the following applications:

Published Applications

Read 234 publications using NB100-142 in the following applications:

Formulation, Preparation, and Storage

Purification

Unpurified

Formulation

Whole antisera

Preservative

0.02% Sodium Azide

Concentration

This product is unpurified. The exact concentration of antibody is not quantifiable.

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: Mre11

Mre11 (double-strand break repair protein MRE11A) is an important component of MRN/X complex (Mre11, Rad50, and Nbs1/Xrs2), playing a central role in double-strand break (DSB) repair, DNA recombination, telomere integrity maintenance, meiotic cell division, and viral infection. Mre11 and Rad50 comprise the catalytic component of the MRN complex with Rad50 involved in ATP hydrolysis and Mre11 functioning as a nuclease having both single-strand endonuclease and double-strand-specific 3'-5' exonuclease activity (1). The MRN complex is also involved in DNA damage signaling via activation of ATM kinase and in telomeres, where it may modulate t-loop formation (2). In addition, Mre11 is a component of the BASC complex (BRCA1, MSH2, MSH6, MLH1, ATM, BLM, RAD50, MRE11A and NBN) and interacts with DCLRE1C/Artemis as well as DCLRE1B/Apollo (3).

The MRN assembles as a hetero-hexamer consisting of 2 RAD50 subunits, 2 MRE11 nucleases, and 2 NBS1 subunits, specific to eukaryotes. Mre11 is highly conserved with the N-terminal region of the human protein sharing approximately 50% amino acid sequence identity with the yeast ortholog. Human Mre11 has 3 known isoforms with the predominant isoform having a theoretical molecular weight of 80.6kDa. This nuclear localized protein has high expression in proliferating tissues. Defects in Mre11 can lead to genomic instability, telomere shortening, aberrant meiosis, hypersensitivity to DNA damage, and ataxia telangiectasia-like disorder (ATLD). MRN expression has been associated with the prognosis of cancer and MRN mutants have been linked to cancer susceptibility in ovarian cancer and glioma (4, 5).

References

1. Paull TT. (2018) 20 Years of Mre11 Biology: No End in Sight. Mol Cell. 71(3):419-427. PMID: 30057197

2. Syed A, Tainer JA. (2018) The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair. Annu Rev Biochem. 87:263-294. PMID: 29709199

3. Wang Y, Cortez D, Yazdi P, Neff N, Elledge SJ, Qin J. (2000) BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev. 14(8):927-39. PMID: 10783165

4. Kim JH, Grosbart M, Anand R, Wyman C, Cejka P, Petrini JHJ. (2017) The Mre11-Nbs1 Interface Is Essential for Viability and Tumor Suppression. Cell Rep. 18(2):496-507. PMID: 28076792

5. Bian L, Meng Y, Zhang M, Li D. (2019) MRE11-RAD50-NBS1 complex alterations and DNA damage response: implications for cancer treatment. Mol Cancer. 18(1):169. PMID: 31767017

Long Name

Meiotic Recombination 11 Homolog A

Alternate Names

MRE11A

Entrez Gene IDs

4361 (Human); 17535 (Mouse)

Gene Symbol

MRE11

UniProt

Product Documents for Mre11 Antibody

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for Mre11 Antibody

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for Mre11 Antibody (NB100-142):

[[URL:https://www.novusbio.com/products/mre11-antibody_nb100-142]][[Caption:M… Antibody]]

Immunoprecipitation Procedure
1. For IP reactions, start with extract (whole cell or nuclear) from around 3 million cells prepared in 0.5-1 ml lysis buffer (100 mM NaCl, 10 mM Tris HCl, 5 mM EDTA, 0.5% nonidet p40).
2. Cells are resuspended in lysis buffer, then incubated with rotation about 15 min at 4 degrees C.
3. The lysate is then centrifuged 5 min at 14000g to remove insoluble material.
4. To cleared lysate, add 1-3 ul of antiserum and incubate on ice for 30 min.
5. Collect immune complexes on Protein A Sepharose by adding 25 ul of a 50% slurry, and incubate with rotation for 1 hour at 4 degrees C.
6. The complexes are pelleted gently (5000g for 5-10 sec.) then washed with 1 ml lysis buffer.
7. Repeat the wash 2 more times.
8. Analyze the immunoprecipitates by SDS PAGE. This antibody works well for IP reactions from both human and mouse cells. The intact complex is stable and can be immunoprecipitated in many common lysis buffers (up to 0.5 M NaCl).
Western Blot Procedure
1. Run 50 ug of protein on a 4-20% Tris-glycine mini-gel at 125V for 90 minutes.
2. Equilibrate gel, nitrocellulose membrane, Whatman paper, and blotting pads in transfer buffer for 15 minutes.
3. Transfer protein to the membrane at 25V for 90 minutes.
4. Allow membrane to air-dry.
5. Block membrane with 1XPBS/3% BSA for 1 hour at room temperature (23-27 degrees C).
6. Wash membrane twice, for 5 minutes each, with 1XPBS/0.05% Tween-20 (PBST).
7. Incubate membrane with 1:5000 dilution of NB100-142 (anti-hMre11), diluted in 1XPBS/1% BSA, for 1 hour at room temperature.
8. Wash membrane once for 15 minutes, then four times for 5 minutes each, with PBST.
9. Incubate membrane with goat anti-rabbit IgG-HRP, diluted in 1XPBS/1% BSA, for 1 hour at room temperature.
10. Wash membrane once for 15 minutes, then four times for 5 minutes each, with PBST.
11. Detect cross-reacting proteins using Renaissance Chemiluminescence Reagent Plus kit from NEN Life Sciences.
NOTE: HeLa whole cell extracts (NB800-PC1) were used as a positive control for this antibody.
Immunofluorescence Procedure
A 5beta in situ extraction method [10mM Pipes, pH 6.8 / 0.2% Triton X-100 / 100mM MgCl2 / 100mM sucrose/ 10mM EGTA Beta on ice] followed by 4% paraformaldehyde fixation of tissues works well for immunofluorescence of anti-hMre11 (NB 100-142).
Please see reference: Franchitto, A., Pichierri, P., Blooms syndrome protein is required for correct relocalization of RAD50/Mre11/nbs1 complex after replication fork arrest. J. of Cell Biology, DOI: 10 (2002)
Immunohistochemistry - FFPE sections
I. Deparaffinization:
A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
B. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
II. Quench Endogenous Peroxidase:
A. Place slides in peroxidase quenching solution: 15-30 minutes.
To Prepare 200 ml of Quenching Solution:
Add 3 ml of 30% Hydrogen Peroxide to 200 ml of Methanol.
Use within 4 hours of preparation
B. Place slides in distilled water: 2 changes for 2 minutes each.
III. Retrieve Epitopes:
A. Preheat Citrate Buffer. Place 200 ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees Celcius.
B. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
C. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
D. Slowly add distilled water to further cool for 5 minutes.E. Rinse slides with distilled water. 2 changes for 2 minutes each.
IV. Immunostaining Procedure:
A. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap Pen).
B. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.
C. Drain wash solution and apply 4 drops of Blocking Reagent to each slide and incubate for 15 minutes.
D. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200 ul of primary antibody solution to each slide, and incubate for 1 hour.
E. Wash slides with Wash Solution: 3 changes for 5 minutes each.
F. Drain wash solution, apply 4 drops of Secondary antibody to each slide and incubate for 1 hour.
G. Wash slides with Wash Solution: 3 changes for 5 minutes each.
H. Drain wash solution, apply 4 drops of DAB Substrate to each slide and develop for 5-10 minutes. Check development with microscope.
I. Wash slides with Wash Solution: 3 changes for 5 minutes each.
J. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
K. Wash slides with Wash Solution: 2-3 changes for 2 minutes each.
L. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
M. Rinse slides in distilled water.
N. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
O. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
P. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
Q. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
R. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.
S. Lay slides on a flat surface to dry prior to viewing under microscope.
NOTES:
Use treated slides (e.g. HistoBond) to assure adherence of FFPE sections to slide.
Prior to deparaffinization, heat slides overnight in a 60 degrees Celcius oven.
All steps in which Xylene is used should be performed in a fume hood.
For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.
For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some
instances, Epitope Retrieval may not be necessary.
200 ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections less than 200 ul may be used.
5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.
Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-1 1/2 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired increase time (up to 10 minutes).

FAQs for Mre11 Antibody

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  • Q: I would like to know if these antibodies are suitable with IHC techniques using HRP conjugated antibodies and DAB as chromogen? Cat# NB200-175, NB100-904 and NB100-142

    A: Yes, all of these antibodies are useful for IHC-P with DAB based detection. Please note that the PTEN Antibody (28H6) NB200-175 has been discontinued, however.

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