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BCL2 Polyclonal Antibody

  • Cat.No.:E-AB-12288

  • Host: Rabbit
  • Reactivity: H
  • Applications: WB,ELISA

To Purchase E-AB-12288

Size:
  • 20μL
  • 60μL
  • 120μL
  • 200μL
Price: $73
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Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (Hela,)

    Western Blot analysis of Hela cells using BCL2 Polyclonal Antibody at dilution of 1:650.

  • Dilution

    WB 1:500-1:1000

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 BCL2 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 0.2 mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 0.05% sodium azide and 50% glycerol, PH7.4
Purification Method Affinity purification
Research Areas Cancer, Cell Biology, Metabolism, Signal Transduction
Conjugation Unconjugated

Immunogen Details

Immunogen Synthetic peptide of human BCL2
Abbre BCL2
Synonyms Apoptosis regulator Bcl 2,Apoptosis regulator Bcl-2,Apoptosis regulator Bcl2,AW986256,B cell CLL/lymphoma 2,B cell leukemia/lymphoma 2,Bcl-2,Bcl2,BCL2,C430015F12Rik,D630044D05Rik,D830018M01Rik,Leukemia/lymphoma,B-cell,2,Oncogene B-cell leukemia 2,PPP1R50,Protein phosphatase 1,regulatory subunit 50
Swissprot P10415
Gene Accession NP_000624
Calculated MW 22kDa
Cellular Localization Mitochondrion outer membrane. Nucleus membrane. Endoplasmic reticulum membrane.
Tissue Specificity Expressed in a variety of tissues.

Background

This gene encodes an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes. Constitutive expression of BCL2, such as in the case of translocation of BCL2 to Ig heavy chain locus, is thought to be the cause of follicular lymphoma. Two transcript variants, produced by alternate splicing, differ in their C-terminal ends.

Citations

  1. ACTA HISTOCHEMICA (2020) IF: 2.107
    Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice

    DOI: 10.1016/j.acthis.2020.151630

    PMID: 32992122

    Sample: Tissue Slice
  2. BIOLOGICAL TRACE ELEMENT RESEARCH (2018) IF: 2.361
    Ethanol via Regulation of NF-κB/p53 Signaling Pathway Increases Manganese-Induced Inflammation and Apoptosis in Hypothalamus of Rats

    DOI: 10.1007/s12011-018-1535-3

    PMID: 30284675

    Sample: Tissue
  3. BMC Complementary and Alternative Medicine (2020) IF: 2.833
    The selective anti-proliferative and pro-apoptotic effect of A. cherimola on MDA-MB-231 breast cancer cell line

    DOI: 10.1186/s12906-020-03120-1

    PMID: 33187495

    Sample: Cell Sample
  4. PHARMACEUTICAL BIOLOGY (2020) IF: 2.971
    Erxian decoction, a famous Chinese medicine formula, antagonizes corticosterone-induced injury in PC12 cells, and improves depression-like behaviours in mice

    DOI: 10.1080/13880209.2020.1765812

    PMID: 32476554

    Sample: Tissue Homogenate
  5. Plants-Basel (2023) IF: 4.658
    The Antioxidant and Proapoptotic Effects of Sternbergia clusiana Bulb Ethanolic Extract on Triple-Negative and Estrogen-Dependent Breast Cancer Cells In Vitro

    DOI: 10.3390/plants12030529

    Sample: Mda-Mb-231 Cell,Mcf-7 Cell
  6. Cancers (2020) IF: 6.126
    Malva pseudolavatera Leaf Extract Promotes ROS Induction Leading to Apoptosis in Acute Myeloid Leukemia Cells In Vitro

    DOI: 10.3390/cancers12020435

    PMID: 32069824

    Sample: Cell Sample
  7. ENVIRONMENTAL POLLUTION (2022) IF: 8.071
    Antioxidative, anti-inflammatory and anti-apoptotic action of ellagic acid against lead acetate induced testicular and hepato-renal oxidative damages and pathophysiological changes in male Long Evans rats

    DOI: 10.1016/j.envpol.2022.119048

    PMID: 35219795

    Sample: Liver,Kidney,Testis
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