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AMPK alpha2 Polyclonal Antibody

Uniprot : Q09137
  • Cat.No.:E-AB-40256

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

To Purchase E-AB-40256

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

Test Application

  • Verified Samples

    Reactivity Application
    Human IHC
    (breast,)

    Immunohistochemistry of paraffin-embedded Human breast using AMPK alpha2 Polyclonal Antibody at dilution of 1:100.

    IF
    (MCF-7,MCF-7,)

    Immunofluorescence analysis of MCF-7 cells using AMPK alpha2 Polyclonal Antibody at dilution of 1:100.

    Immunofluorescence analysis of MCF-7 cells using AMPK alpha2 Polyclonal Antibody at dilution of 1:100.

    Rat WB
    (heart,muscle,)

    Western Blot analysis of Rat heart and Rat muscle using AMPK alpha2 Polyclonal Antibody at dilution of 1:1000.

    Western Blot analysis of Rat heart and Rat muscle using AMPK alpha2 Polyclonal Antibody at dilution of 1:1000.

  • Dilution

    WB 1:500-1:2000 IHC 1:100-1:200 IF 1:100-1:400

  • 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 gel10%
    Blocking1.5 h
    Primary Antibody1:1000
    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 10% 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 AMPK alpha2 Antibody at 1:1000, 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 1 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, Signal Transduction
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant Rat 5'-AMP-activated protein kinase catalytic subunit alpha-2 protein(PKSR040467)
Abbre AMPK alpha2
Synonyms 5'-AMP-activated protein kinase catalytic subunit alpha-2,AAPK2,ACACA kinase,Acetyl-CoA carboxylase kinase,AMPK alpha 2 chain,AMPK subunit alpha-2,AMPK2,AMPKa2,AMPKalpha2,HMGCR kinase,Hydroxymethylglutaryl-CoA reductase kinase,PRKAA,PRKAA2,Protein kinase AMP activated alpha 2 catalytic subunit,Protein kinase AMP activated catalytic subunit alpha 2
Swissprot Q09137
Gene ID 78975
Calculated MW 62,4 kDa
Observed MW 62 kDa

Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. The common factors include:

1. Post-translational modifications: For example, modifications such as glycosylation, phosphorylation, methylation, and acetylation will increase the molecular weight of the protein.

2. Splicing variants: Different expression patterns of various mRNA splicing bodies may produce proteins of different sizes.

3. Post-translational cleavage: Many proteins are first synthesized into precursor proteins and then cleaved to form active forms, such as COL1A1.

4. Relative charge: the composition of amino acids (the proportion of charged amino acids and uncharged amino acids).

5. Formation of multimers: For example, in protein dimer, strong interactions between proteins can cause the bands to be larger. However, the use of reducing conditions can usually avoid the formation of multimers.

If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane.

Cellular Localization Cytoplasm. Nucleus.
Tissue Specificity Skeletal muscle, lower levels in liver, heart and kidney.

Background

The protein encoded by this gene is a catalytic subunit of the AMP-activated protein kinase (AMPK). AMPK is a heterotrimer consisting of an alpha catalytic subunit, and non-catalytic beta and gamma subunits. AMPK is an important energy-sensing enzyme that monitors cellular energy status. In response to cellular metabolic stresses, AMPK is activated, and thus phosphorylates and inactivates acetyl-CoA carboxylase (ACC) and beta-hydroxy beta-methylglutaryl-CoA reductase (HMGCR), key enzymes involved in regulating de novo biosynthesis of fatty acid and cholesterol. Studies of the mouse counterpart suggest that this catalytic subunit may control whole-body insulin sensitivity and is necessary for maintaining myocardial energy homeostasis during ischemia.

Reviews/Q&A

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  • Reviews
  • Q&A

Verified Customer

B***nSubmitted [ Oct 25 2019 ]

  • Application:WB
  • Species:Rat
  • Loading amount:50μg
  • Sample source:Rat
  • Gel Running Conditions:Reduced, Denaturing, Other details:12
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 2hours

  • Primary antibody:

    Dilution:1:1000

    Time: 15hours

  • Temperature:4℃

    Diluent:5%non-fat milk

  • Secondary antibody:Use Elabscience secondary antibody Goat anti rabbit
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Pretty good.No unspecific band was detected.
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Verified Customer

B***nSubmitted [ May 30 2019 ]

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

    Blocking concentration:10 

    Blocking temperature:37℃

    Blocking time: 30minutes

  • Primary antibody:Undiluted

    Time: 17hours

  • Temperature:4℃

    Diluent:PBS

  • Secondary antibody:Use Non-Elabscience secondary antibody Goat Anti-Rabbit IgG (H+L)(peroxidase/HRP(Goat,HRP)
  • Dilution:1:200
  • Description:AMPK alpha2 presents in both the nuclear and the cytoplasm in human breast cancer,The signal is very clear and accuracy.
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Verified Customer

J***ySubmitted [ May 29 2019 ]

  • Application:IHC
  • Species:Human
  • Sample source:Human Kidney
  • Blocking:

    Blocking concentration:10 

    Blocking temperature:37℃

    Blocking time: 30minutes

  • Primary antibody:Undiluted

    Time: 15hours

  • Temperature:4℃

    Diluent:PBS

  • Secondary antibody:Use Non-Elabscience secondary antibody Anti-rabbit-HRP(Goat,Horse Radish Peroxidase)
  • Description:Both cytoplasm and nucleus were expressed in normal human kidney tissues
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