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Anti-BRE Rabbit Antibody [C2G22]

Catalog No.: F1273

Application: Reactivity: Human, Mouse, Rat, Monkey

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human, Mouse, Rat, Monkey

Source
Rabbit

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
45 kDa

Positive Control	HeLa cell; NIH/3T3 cell; HepG2 cell; 293T cell; COS-7 cell; MTLn3 cell
Negative Control

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
BRE Rabbit mAb recognizes endogenous levels of total BRE protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q9NXR7

Clone
C2G22

Synonym(s)
BRISC and BRCA1-A complex member 2 , BRCA1-A complex subunit BRE, BRCA1/BRCA2-containing complex subunit 45, Brain and reproductive organ-expressed protein, BABAM2, BRCC45, BRE

Background
BRE (Brain and reproductive organ-expressed protein), also known as BRCC45, is a multifunctional protein found in both the cytoplasm and nucleus, encoded by a stress-responsive gene producing multiple mRNA isoforms. Structurally, BRE is a component of the BRCA1-A complex, where it interacts with proteins like MERIT40 to facilitate DNA damage repair through homologous recombination. Functionally, BRE plays key roles in DNA repair, apoptosis inhibition, cell differentiation, and tissue repair; it contributes to genomic stability by assisting in the repair of double-strand DNA breaks and preventing premature cellular senescence. In the nucleus, BRE functions as a critical component of the BRCA1-A complex, interacting with MERIT40 to maintain complex integrity and facilitate DNA damage repair through homologous recombination by supporting BRCC36’s K63 deubiquitinase activity and enhancing BRCA1-BARD1 E3 ligase function. In the cytoplasm, BRE is part of the BRISC complex, regulating K63-linked polyubiquitin cleavage, and acts as an anti-apoptotic factor by binding to Fas, TNF-R1, and the death-inducing signaling complex to inhibit apoptosis. BRE is widely expressed across tissues and is dynamically regulated in response to cellular stressors such as ionizing radiation and hormonal signals. Its expression is frequently upregulated in cancers like esophageal squamous cell carcinoma (ESCC), liver, and lung cancers, where it promotes tumor growth and survival, often through activation of pro-survival pathways like AKT signaling, though its role in tumorigenesis may vary by cancer type.

Background

BRE (Brain and reproductive organ-expressed protein), also known as BRCC45, is a multifunctional protein found in both the cytoplasm and nucleus, encoded by a stress-responsive gene producing multiple mRNA isoforms. Structurally, BRE is a component of the BRCA1-A complex, where it interacts with proteins like MERIT40 to facilitate DNA damage repair through homologous recombination. Functionally, BRE plays key roles in DNA repair, apoptosis inhibition, cell differentiation, and tissue repair; it contributes to genomic stability by assisting in the repair of double-strand DNA breaks and preventing premature cellular senescence. In the nucleus, BRE functions as a critical component of the BRCA1-A complex, interacting with MERIT40 to maintain complex integrity and facilitate DNA damage repair through homologous recombination by supporting BRCC36’s K63 deubiquitinase activity and enhancing BRCA1-BARD1 E3 ligase function. In the cytoplasm, BRE is part of the BRISC complex, regulating K63-linked polyubiquitin cleavage, and acts as an anti-apoptotic factor by binding to Fas, TNF-R1, and the death-inducing signaling complex to inhibit apoptosis. BRE is widely expressed across tissues and is dynamically regulated in response to cellular stressors such as ionizing radiation and hormonal signals. Its expression is frequently upregulated in cancers like esophageal squamous cell carcinoma (ESCC), liver, and lung cancers, where it promotes tumor growth and survival, often through activation of pro-survival pathways like AKT signaling, though its role in tumorigenesis may vary by cancer type.

References
https://pubmed.ncbi.nlm.nih.gov/32850455/ https://pubmed.ncbi.nlm.nih.gov/27001068/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%