Click here to view cell apoptosis assay products

BRCA1 Polyclonal Antibody

Uniprot : P38398
  • Cat.No.:E-AB-40282

  • Host: Rabbit
  • Reactivity: H,M
  • Applications: IHC,IF

To Purchase E-AB-40282

Size:
  • 20μL
  • 60μL
  • 120μL
  • 200μL
Price: $65
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human IHC
    (breast cancer,)

    Immunohistochemistry of paraffin-embedded Human breast cancer using BRCA1 Polyclonal Antibody at dilution of 1:100.

    IF
    (MCF-7,MCF-7,U-2OS,U-2OS,)

    Immunofluorescence analysis of MCF-7 cells using BRCA1 Polyclonal Antibody at dilution of 1:100.

    Immunofluorescence analysis of MCF-7 cells using BRCA1 Polyclonal Antibody at dilution of 1:100.

    Immunofluorescence analysis of U-2OS cells using BRCA1 Polyclonal Antibody at dilution of 1:100.

    Immunofluorescence analysis of U-2OS cells using BRCA1 Polyclonal Antibody at dilution of 1:100.

    Mouse IHC
    (brain,)

    Immunohistochemistry of paraffin-embedded Mouse brain using BRCA1 Polyclonal Antibody at dilution of 1:100.

  • Dilution

    IHC 1:100-1:200 IF 1:100-1:400

Preparation of protein samples

1.Protein extraction

1)For tissue sample
a. Take the samples, wash the tissue thoroughly with pre-cooled PBS (0.01 M, pH=7.4)(Cat# E-BC-R187) to remove the surface blood and internal debris.
b. Weigh and smash the tissue, add an appropriate ratio of RIPA Lysis Buffer (Cat# E-BC-R327)(add 10 μL PMSF and 10 μL Na3VO4 to each 1 mL RIPA Lysis) and homogenizely lyse the tissue.
It is recommended to homogenize according to the ratio of tissue weight: RIPA volume = 3:10. For example, add 1 mL RIPA Lysis Buffer to 0.3 g tissue sample, the specific volume can be adjusted according to experimental requirements.
c. Shake and lyse on the ice for 30 min after homogenization. And then sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lysis and reduce the viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2)For cell sample
a. Collect the cells, wash them thoroughly with pre-cooled PBS (0.01 M, pH=7.4) to remove the medium off (it is generally recommended to wash 3 times).
b. Add an appropriate ratio of RIPA Lysate Buffer (10 μL PMSF and 10 μL Na3VO4 in each 1 mL RIPA Lysis) and lyse on the ice for 30 min.
It is recommended to add 0.1 mL of RIPA Lysis Buffer to each well of a 6-well plates (the protein content in different cells may vary, and the volume of the lysate added can be appropriately adjusted).
c. Sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lyse and reduce viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2.Measurement of protein concentration
By the BCA method (see the Total Protein Colorimetric Assay Kit (Cat# E-BC-K318) instructions).

3.Boiling the samples
Adjust the protein concentration with PBS Buffer. Add 5 × SDS Loading Buffer (Cat# E-BC-R288) with the ratio of the protein sample: 5 × SDS Loading Buffer = 4:1 and boil the mixture for 10 min. Centrifuge at 12,000 rpm for 2 min and collect the supernatant. The denatured protein can be employed to Western Blot experiments or stored at -20℃ or -80℃.

Note: It is recommended that the total protein loading amount of test sample is about 50 μg in each well. Try to make the loading volume of each sample close to 10 μL.

Electrophoresis

1.According to the molecular weight of the target protein, prepare 0% separation gel. Add the test sample to each well, and add 5 μL of Pre-stained Protein Marker (Cat# E-BC-R273)to a reserved well in order to verify the target molecular weight and the extent of membrane transfer. Add Electrophoresis Buffer ( Cat# E-BC-R331) and start electrophoresis.

2.Electrophoresis at 80v when the samples are in stacking gel, then convert to 120v when the blue flow into the separating gel. Electrophoresis time is about 2-3 h till bromophenol blue reaches the bottom of the gel.

Transfer Membrane

1.Choose the PVDF Membrane (Cat# E-BC-R266) with a pore size of μm according to the molecular weight of the target protein. Soak the PVDF Membrane in methanol for 1 min to activate it, and then soak the PVDF Membrane in the Transmembrane Buffer (Cat# E-BC-R333), the filter paper and fiber mat must be soaked in the Transmembrane Buffer for use too.

2.Follow manufacture instructions of Transfer System for wet, semi-dry, or dry transfer.

Incubation of antibodies

1.Soak the PVDF Membrane with TBST Buffer (Cat# E-BC-R335) containing 5% Skim Milk Powder as blocking buffer and block the membrane at room temperature for .

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the BRCA1 Antibody at , soak the PVDF Membrane in the primary antibody working solution, incubate overnight at 4 ℃, and gently shake.

3.Wash the PVDF Membrane with TBST Buffer for .

4.According to the recommended secondary antibody dilution ratio, use a TBST Buffer solution containing 2% Skim Milk Powder to dilute Goat Anti-Rabbit IgG (H+L) (peroxidase/HRP conjugated) (Cat# E-AB-1003) at . Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for .

Detection

1.Mix A and B in the Excellent Chemiluminescent Substrate Detection kit (Cat# E-BC-R347) at the ratio of 1:1 as working solution.

2.Take out the PVDF Membrane from TBST Buffer and absorb the liquid with the filter paper. Pave the PVDF Membrane on the detection machine, add ECL working solution continuously on the PVDF Membrane, discharge the bubble and detect the result.

3.Adjust the contrast and the exposure time to get the best image.

Appendix

Product Details

Clonality Polyclonal
Isotype IgG
Concentration 0.6 mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 0.05% Proclin300 and 50% glycerol, pH7.4.
Purification Method Antigen Affinity Purification
Research Areas Cancer, Cell Biology, Epigenetics and Nuclear Signaling
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant Human Breast cancer type 1 susceptibility protein(PKSH033832)
Abbre BRCA1
Synonyms BRCA 1, BRCA1, BRCA1 DNA repair associated, BRCA1/BRCA2 containing complex subunit 1, BRCAI, BRCC 1, BRCC1, Breast and ovarian cancer susceptibility protein 1, Breast Cancer 1, BROVCA1, FANCS, IRIS, PNCA4, PPP1R53
Swissprot P38398
Gene ID 672
Cellular Localization Nucleus
Tissue Specificity Isoform 1 and isoform 3 are widely expressed. Isoform 3 is reduced or absent in several breast and ovarian cancer cell lines.

Background

RCA1,also named as RNF53,plays a central role in DNA repair by facilitating cellular response to DNA repair. It is required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. The BRCA1-BARD1 heterodimer coordinates a diverse range of cellular pathways such as DNA damage repair,ubiquitination and transcriptional regulation to maintain genomic stability. BRCA1 acts by mediating ubiquitin E3 ligase activity that is required for its tumor suppressor function. It is involved in transcriptional regulation of P21 in response to DNA damage. BRCA1 is required for FANCD2 targeting to sites of DNA damage. It may function as a transcriptional regulator. BRCA1 inhibits lipid synthesis by binding to inactive phosphorylated ACACA and preventing its dephosphorylation. The antibody is specific to BRCA1. BRCA1 appears to produce multiple splice variants. BRCA1 is a nuclear protein with a molecular mass of 220 kDa. The present study describes the isolation and expression of two cDNAs of BRCA1,including a splice variant designated BRCA1D672-4095. BRCA1D672-4095 is generated by exclusion of exon 11 by in-frame splicing and produces a 97 kDa protein. In contrast to BRCA1,BRCA1D672-4095 localizes to the cytoplasm.

Reviews/Q&A

  • Show all
  • Reviews
  • Q&A

Verified Customer

l******mSubmitted [ Dec 20 2019 ]

  • Application:IHC
  • Species:Human
  • Sample source:Human breast cancer tissue
  • Blocking:

    Blocking buffer:BSA

    Blocking concentration:10 %

    Blocking temperature:37℃

    Blocking time: 0.5hours

  • Primary antibody:

    Dilution:1:100

    Time: 2hours

  • Temperature:37℃

  • Secondary antibody:Use Elabscience secondary antibody E-IR-R213
Read More
Read Less
... Show All Show Less

People Also Bought

Apply for 10 Test Free Trial FCM Antibody
*Product Name:
*Catalog Number:
*Name:
*Email:
*Message:
*Country:
*When will you use it?

*Captcha: