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ADIPOQ Polyclonal Antibody

Uniprot : Q60994
  • Cat.No.:E-AB-40301

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

To Purchase E-AB-40301

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

Test Application

  • Verified Samples

    Reactivity Application
    Human IHC
    (liver cancer,)

    Immunohistochemistry of paraffin-embedded Human liver cancer using ADIPOQ Polyclonal Antibody at dilution of 1:200.

    Mouse WB
    (heart,)

    Western Blot analysis of Mouse heart using ADIPOQ Polyclonal Antibody at dilution of 1:500.

  • Dilution

    WB 1:500-1:1000 IHC 1:100-1:300

  • Western Blot Operation Guide

    In order to facilitate the operation and ensure the accuracy of WB results, the Western Blot Detection kit (Cat# E-IR-R304) is now available, containing the reagents which are needed from sample preparation to result detection. Please order the appropriate kit according to your specific needs.

    Separating gel12%
    Blocking1.5 h
    Primary Antibody1:500
    Secondary Antibody1:5000
    Click Here for More Details .. More ↓

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 12% 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 0.45 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 1.5 h.

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the ADIPOQ Antibody at 1:500, 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 3 times, 15 min/time.

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 1:5000. Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for 3 times, 15 min/time.

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 0.02 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, Cardiovascular, Metabolism, Neuroscience, Signal Transduction, Stem Cells
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant Mouse Adiponectin protein(PKSM500009)
Abbre ADIPOQ
Synonyms 30 kDa adipocyte complement related protein,30 kDa adipocyte complement-related protein,ACDC,ACRP30,ADIPO,Adipocyte,Adipocyte C1q and collagen domain containing protein,Adipocyte complement related 30 kDa protein,Adipocyte complement related protein of 30 kDa,Adipocyte complement-related 30 kDa protein,adipocyte-specific secretory protein,Adiponectin,Adiponectin precursor,adiponectin,C1Q and collagen domain containing,Adipoq,Adipose most abundant gene transcript 1,Adipose most abundant gene transcript 1 protein,Adipose specific collagen like factor,ADIPQTL1,ADPN,APM 1,apM-1,APM1,C1q and collagen domain-containing protein,GBP28,Gelatin binding protein,Gelatin binding protein 28,Gelatin-binding protein
Swissprot Q60994
Gene ID 11450
Calculated MW 27kDa
Observed MW 27kDa
Cellular Localization Secreted.
Tissue Specificity Synthesized exclusively by adipocytes and secreted into plasma.Highest expression level in subcutaneous adipose tissue.

Background

Adiponectin (AdipoQ),an adipocyte-derived hormone,is one of the most abundant adipokines in the blood circulation. Adiponectin modulates a number of metabolic processes,including improving insulin sensitivity and anti-inflammatory activity. The role of AdipoQ in reproduction is not yet fully understood,but the expression of AdipoQ in reproductive tissues has been observed in various animals and humans,including chicken testis,bovine ovary,and human placenta. Adiponectin exerts its effects by activating a range of different signaling molecules via binding to two transmembrane AdipoQ receptors,AdipoR1 and AdipoR2. AdipoR1 is expressed primarily in the skeletal muscle,whereas AdipoR2 is predominantly expressed in the liver. AdipoQ May play a role in cell growth,angiogenesis and tissue remodeling by binding and sequestering various growth factors.

Reviews/Q&A

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  • Reviews
  • Q&A

Verified Customer

P****pSubmitted [ Nov 13 2019 ]

  • Application:WB
  • Species:Mouse
  • Loading amount:50μg
  • Sample source:mouse heart
  • Gel Running Conditions:Reduced, Denaturing, Other details:15
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1.5hours

  • Primary antibody:

    Dilution:1:500

    Time: overnhours

  • Temperature:4℃

    Diluent:5% skim milk

  • Secondary antibody:Use Non-Elabscience secondary antibody Goat anti rabbit(,HRP)
  • Dilution:1:10000
  • Detection:

    method:ECL

  • Description:Excellent product. single specific band around 27Kda.
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Verified Customer

I*****nSubmitted [ Sep 20 2019 ]

  • Application:IHC
  • Species:Mouse
  • Sample source:skin
  • Blocking:

    Blocking buffer:Serum

    Blocking concentration:10 %

    Blocking temperature:25℃

    Blocking time: 1hours

  • Primary antibody:

    Dilution:1:200

    Time: 18hours

  • Temperature:4℃

    Diluent:PBS

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