BRCA2 antibody (AA 21-130)

Catalog No. ABIN673434

This anti-BRCA2 antibody is a Rabbit Polyclonal antibody detecting BRCA2 in WB, ELISA, IHC (p), IHC (fro), IF (cc) and IF (p). Suitable for Human. Independently validated for use in Western Blotting.

Quick Overview for BRCA2 antibody (AA 21-130) (ABIN673434)

Target: BRCA2 (Breast Cancer 2, Early Onset (BRCA2))

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This BRCA2 antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p))

Product Details anti-BRCA2 Antibody

Binding Specificity: AA 21-130

Cross-Reactivity: Human

Predicted Reactivity: Mouse,Rat,Dog,Cow,Horse,Chicken,Rabbit

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human BRCA2

Isotype: IgG

Application Details

Application Notes: WB 1:300-5000
ELISA 1:500-1000
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Restrictions: For Research Use only

Independent Validation (WB)

Validation #029807 (Western Blotting)

by: Alamo Laboratories Inc

No.: #029807

Date: 08/26/2014

Lot Number: 101020

Method validated: Western Blotting

Positive Control: MCF7 cells

Negative Control: [CAPAN1 cells] (http://www.pnas.org/content/98/15/8644.full.pdf)

Notes: A strong band was observed in the positive control sample at the correct molecular weight. No bands were observed in the negative control sample.

Validation Images

Figure 1: Western Blot for BRCA2. Arrowhead indicates the expected molecular weight of ~384 kDa.

Full Methods

Primary Antibody:

• Antigen: Breast Cancer 2, Early Onset (BRCA2)
• Catalog number: ABIN673434
• Lot number: 101020
• Antibody Dilution: 1:200

Secondary Antibody:

• Antigen: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate
• Lot number: L170-6515
• Antibody Dilution: 1:10,000

Full Protocol:

• 1. The cell extracts were heated at 95°C for 5 minutes in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol.
• 2. 15 μl of heated culture-media were loaded and resolved on 8-16% SDS-polyacrylamide gel.
• 3. The Thermo Scientific - Spectra Multicolor Broad Range (Cat # 26634) were used as molecular mass markers.
• 4. Proteins were then transferred onto PVDF membrane by wet transfer and protein transfer was confirmed with Ponceau-S staining.
• 5. The PVDF membrane was incubated with 25 ml of blocking buffer [Tris Buffered Saline, pH 7.4 plus 0.1% TW20 (TBST)] containing 5% (W/V) BSA at room temperature for 1 hour.
• 6. The membrane was rinsed with TBST once.
• 7. The membrane was immersed with the protein side up in the primary antibody solution in TBST containing 5% (W/V) BSA and incubated for 10 hours at 4°C.
• 8. The membrane was rinsed in TBST thrice for 5 minutes each.
• 9. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBST containing 5% (W/V) BSA and incubated for 1 hour at room temperature (~26°C) with gentle agitation.
• 10. The membrane was rinsed thrice TBST thrice for 5 minutes each.
• 11. The membrane was rinsed in TBS twice for 30 seconds each.
• 12. Signals were detected with ECL-2 Substrate. The blot was scanned for 5 minutes.
• 13. The membrane was rinsed three times TBST.
• 14. Incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 minutes each.
• 15. The membrane was washed in TBST 2 times for 10 minutes each.
• 16. Repeated Steps 5-12 with the loading control antibody (for Anti-actin) and its matching secondary antibody.

Experimental Notes: - No experimental challenges noted.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

Target Details for BRCA2

Target: BRCA2 (Breast Cancer 2, Early Onset (BRCA2))

Alternative Name: BRCA2

Background:

Synonyms: FAD, FACD, FAD1, GLM3, BRCC2, FANCD, PNCA2, FANCD1, BROVCA2, Breast cancer type 2 susceptibility protein, Fanconi anemia group D1 protein, BRCA2

Background: Involved in double-strand break repair and/or homologous recombination. Binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). Acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. Part of a PALB2-scaffolded HR complex containing RAD51C and which is thought to play a role in DNA repair by HR. May participate in S phase checkpoint activation. Binds selectively to ssDNA, and to ssDNA in tailed duplexes and replication fork structures. May play a role in the extension step after strand invasion at replication-dependent DNA double-strand breaks, together with PALB2 is involved in both POLH localization at collapsed replication forks and DNA polymerization activity. In concert with NPM1, regulates centrosome duplication. Interacts with the TREX-2 complex (transcription and export complex 2) subunits PCID2 and DSS1, and is required to prevent R-loop-associated DNA damage and thus transcription-associated genomic instability. Silencing of BRCA2 promotes R-loop accumulation at actively transcribed genes in replicating and non-replicating cells, suggesting that BRCA2 mediates the control of R-loop associated genomic instability, independently of its known role in homologous recombination (PubMed:24896180).

Gene ID: 675

UniProt: P51587

Pathways: DNA Damage Repair, M Phase, Maintenance of Protein Location