Bcl-2 Polyclonal Antibody

  • Cat.No.:E-AB-60012

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

To Purchase E-AB-60012

Size:
  • 60μL
  • 120μL
  • 200μL
Price: $129
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Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (HL-60,HL-60,)

    Western blot analysis of extracts of HL-60 cells using Bcl-2 Polyclonal Antibody at dilution of 1:500.

    Western blot analysis of extracts of various cells using Bcl-2 Polyclonal Antibody at dilution of 1:1000.

    IHC
    (lung cancer,)

    Immunohistochemistry of paraffin-embedded Human lung cancer using Bcl-2 Polyclonal Antibody at dilution of 1:100 (40x lens).

    IF
    (U-2OS,)

    Immunofluorescence analysis of U-2OS cells using Bcl-2 Polyclonal Antibody at dilution of 1:100 (40x lens).Blue: DAPI for nuclear staining.

    Rat WB
    (lung,liver,)

    Western blot analysis of extracts of various cells using Bcl-2 Polyclonal Antibody at dilution of 1:1000.

    Western blot analysis of extracts of various cells using Bcl-2 Polyclonal Antibody at dilution of 1:1000.

    IHC
    (brain,)

    Immunohistochemistry of paraffin-embedded Rat brain using Bcl-2 Polyclonal Antibody at dilution of 1:100 (40x lens).

    IF
    (C6,)

    Immunofluorescence analysis of C6 cells using Bcl-2 Polyclonal Antibody at dilution of 1:100 (40x lens).Blue: DAPI for nuclear staining.

    Mouse WB
    (testis,)

    Western blot analysis of extracts of various cells using Bcl-2 Polyclonal Antibody at dilution of 1:1000.

    IHC
    (testis,)

    Immunohistochemistry of paraffin-embedded Mouse testis using Bcl-2 Polyclonal Antibody at dilution of 1:100 (40x lens).

    IF
    (NIH/3T3,)

    Immunofluorescence analysis of NIH/3T3 cells using Bcl-2 Polyclonal Antibody at dilution of 1:100 (40x lens).Blue: DAPI for nuclear staining.

  • Dilution

    WB 1:500-1:2000 IHC 1:50-1:100 IF 1:50-1:100

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 (Wet transfer)

1.Choose the PVDF Membrane (Cat# ) 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.Place the following materials in the order of the black plate (negative electrode) - fiber mat - filter paper - gel - PVDF Membrane - filter paper - fiber mat - white plate (positive electrode) are placed in order, discharge bubbles, clamp and place in the wet transfer tank. The recommended transmembrane conditions are . Make sure that the transmembrane process is carried out at low temperatures.
Note: This is for wet transfer. If other transmembrane methods are used, please adjust according to the specific conditions.

3.After the transmembrane, take out the PVDF Membrane carefully and wash with TBST Buffer for 1 min.

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 Bcl-2 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

Isotype IgG
Concentration 1mg/mL
Storage Store at -20°C. Avoid freeze / thaw cycles.
Buffer PBS with 0.02% sodium azide, 50% glycerol, pH7.3.
Purification Method Affinity purification
Research Areas Cancer, Cell Biology, Metabolism, Signal Transduction
Conjugation Unconjugated

Immunogen Details

Immunogen A synthetic peptide of human Bcl-2
Abbre Bcl-2
Synonyms Bcl-2,PPP1R50,BCL2
Swissprot P10415
Gene ID 596
Calculated MW 22kDa/26kDa
Observed MW 24kDa/26kDa
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. Alternative splicing results in multiple transcript variants.

Citations

From now on, if you have published a paper by using any of our products since 1/1/2019, fill out the “Elabscience Publication Reward Application Form”carefully and send it to orders@elabscience.com, we will get back to you with the reward after we confirm it ASAP!

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  1. Toxicology (2019) IF: 3.265
    Ethanol increases manganese-Induced spatial learning and memory deficits via oxidative/nitrosative stress induced p53 dependent/independent hippocampal apoptosis

    DOI: 10.1016/j.tox.2019.03.001

    PMID: 30844427

    Sample: Tissue homogenate
  2. Biochemical and Biophysical Research Communications (2020) IF: 2.985
    Regulated rutin co-administration reverses mitochondrial-mediated apoptosis in Plasmodium berghei-infected mice

    DOI: 10.1016/j.bbrc.2019.11.067

    PMID: 31767147

    Sample: Tissue homogenate
  3. BMC complementary and alternative medicine (2019) IF: 2.833
    The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines

    DOI: 10.1186/s12906-019-2768-1

    PMID: 31830975

    Sample: Cell culture supernatant
  4. Biological trace element research (2018) IF: 2.361
    Ethanol Exacerbates Manganese-Induced Neurobehavioral Deficits, Striatal Oxidative Stress, and Apoptosis Via Regulation of p53, Caspase-3, and Bax/Bcl-2 Ratio-Dependent Pathway

    DOI: 10.1007/s12011-018-1587-4

    PMID: 30488170

    Sample: Tissue homogenate
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