Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875)

Overview

  • Product name
    Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody
  • Description
    Rabbit polyclonal to AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172)
  • Specificity
    ab23875 recognises the phosphorylated forms of AMPK alpha 1 (T172) and AMPK alpha 2 (T183).
  • Tested applications
    Suitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human AMPK alpha 1 (phospho T183). Also within AMPK alpha 2 (phospho T172).

  • Positive control
    • Insulin treated CHO T cells, Insulin treated 3T3L1, Metformin treated L6 myoblast cells. Metformin treated HepG2 cells (10 mM for 24 hr).

Properties

Applications

Our Abpromise guarantee covers the use of ab23875 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use at an assay dependent concentration. Predicted molecular weight: 62 kDa.
IHC-P 1/10 - 1/50.
Flow Cyt 1/20.
ICC/IF 1/250.

Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody images

  • Immunocytochemistry/ Immunofluorescence analysis of 70% confluent log phase MDA-MB-231 cells labeling AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) with ab23875. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875) at 1ug/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with mountant with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin, 1/300. Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

  • All lanes : Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875)

    Lane 1 : Lysates prepared from HepG2 cells left unstimulated with 3% BSA-TBST buffer and no peptide
    Lane 2 : Lysates prepared from HepG2 cells stimulated with Metformin with 3% BSA-TBST buffer and no peptide
    Lane 3 : Lysates prepared from HepG2 cells stimulated with Metformin with 3% BSA-TBST buffer and the non-phosphopeptide corresponding to the immunogen
    Lane 4 : Lysates prepared from HepG2 cells stimulated with Metformin with 3% BSA-TBST buffer and a generic phospho-threonine-containing peptide
    Lane 5 : Lysates prepared from HepG2 cells stimulated with Metformin with 3% BSA-TBST buffer and the phosphopeptide immunogen
    Lane 6 : Lysates prepared from HepG2 cells stimulated with Metformin and treated with lambda phosphatase with 3% BSA-TBST buffer

    Secondary
    goat F(ab’)2 anti rabbit IgG HRP conjugate

    Predicted band size : 62 kDa
    Observed band size : 60 kDa (why is the actual band size different from the predicted?)


    Exposure time : 2 hours

    Lysates were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF, treated or not with lambda phosphatase, blocked with a 3% BSA-TBST buffer for one hour at room temperature, incubated with relevant peptides (see below) and incubated with the AMPK alpha 1/2 [pT 172] antibody for two hours at room temperature in 3% BSA-TBST buffer.

    After washing, membranes were incubated with goat F(ab’)2 antirabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal™ method.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue sections labeling AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) with ab23875 (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875) diluted in 3% BSA-PBS at a dilution of 1/20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Flow Cytometry analysis of MDA-MB-231 cells labeling AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) with ab23875. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875, red) or with rabbit isotype control (pink) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

References for Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) antibody (ab23875)

ab23875 has not yet been referenced specifically in any publications.

Product Wall

Application
ChIP
Sample
Mouse Tissue lysate - nuclear (liver)
Negative control
IgG antibodies
Specification
liver
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Positive control
antibodies from the article - PMC5159533 "Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARa"
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Verified customer

Submitted Oct 17 2017

The concentration of ab23875 lot GR3189992-1 is 0.75 mg/ml.

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain Tissue)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer
Specification
Brain Tissue
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde
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Submitted Oct 26 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - 0.2% Triton X100.2% Triton X100
Specification
Brain
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

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Verified customer

Submitted Sep 21 2016

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this h...

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I have been informed that to detect phospho AMPK, metformin was used at 10 mM for 24 hr in treating HepG2 cells. I was unable to have further information regarding the medium unfortunately, but I hope the above information will help you,

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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