Overview

  • Product name
    Human AMPK alpha 1 ELISA Kit
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    HEK293T 8 7%
    Inter-assay
    Sample n Mean SD CV%
    HEK293T 3 11%
  • Sample type
    Cell culture extracts, Adherent cells, Suspension cells, Tissue Extracts
  • Assay type
    Sandwich (quantitative)
  • Sensitivity
    0.452 ng/ml
  • Range
    1.6 ng/ml - 100 ng/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture media 98 90% - 105%

  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    Abcam’s AMPK-alpha 1 in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of AMPK-alpha 1 protein in cell and tissue extracts.

    The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

     

  • Notes

    AMP-activated protein kinase (AMPK) is an energy sensor protein kinase that plays a key role in regulating cellular energy homeostasis. Mammalian AMPK is a heterotrimer kinase, containing a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). Each subunit has different isoforms (alpha 1, alpha 2, beta 1, beta 2, gamma 1, gamma 2, gamma 3) with differential tissue expression, cellular localization and functionality. Human AMPK has a 98.7% sequence similarity with mouse AMPK and a 99% sequence similarity with rat AMPK. It has been hypothesized that when ADP or AMP are present at high levels, these nucleotides bind directly to the gamma subunit, leading to a conformational change that allows phosphorylation of Thr172 at the alpha subunit. Phosphorylation of AMPK alpha activates the kinase, which leads to downstream effects concerted to increase catabolic pathway and suppress anabolic pathways in order to restore levels of cellular ATP and ultimately control cell fate.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X AMPK-alpha Capture Antibody 1 x 600µl
    10X AMPK-alpha Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml
    5X Cell Extraction Buffer PTR 1 x 10ml
    AMPK-alpha Human Lyophilized Recombinant Protein 2 vials
    Antibody Diluent 5BI 1 x 6ml
    Plate Seals 1 unit
    Sample Diluent NS (ab193972) 1 x 12ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Substrate 1 x 12ml
  • Research areas
  • Relevance
    Catalytic subunit of AMP-activated protein kinase (AMPK), an energy sensor protein kinase that plays a key role in regulating cellular energy metabolism. In response to reduction of intracellular ATP levels, AMPK activates energy-producing pathways and inhibits energy-consuming processes: inhibits protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation. AMPK acts via direct phosphorylation of metabolic enzymes, and by longer-term effects via phosphorylation of transcription regulators. Also acts as a regulator of cellular polarity by remodeling the actin cytoskeleton; probably by indirectly activating myosin. Regulates lipid synthesis by phosphorylating and inactivating lipid metabolic enzymes such as ACACA, ACACB, GYS1, HMGCR and LIPE; regulates fatty acid and cholesterol synthesis by phosphorylating acetyl-CoA carboxylase (ACACA and ACACB) and hormone-sensitive lipase (LIPE) enzymes, respectively. Regulates insulin-signaling and glycolysis by phosphorylating IRS1, PFKFB2 and PFKFB3. AMPK stimulates glucose uptake in muscle by increasing the translocation of the glucose transporter SLC2A4/GLUT4 to the plasma membrane, possibly by mediating phosphorylation of TBC1D4/AS160. Regulates transcription and chromatin structure by phosphorylating transcription regulators involved in energy metabolism such as CRTC2/TORC2, FOXO3, histone H2B, HDAC5, MEF2C, MLXIPL/ChREBP, EP300, HNF4A, p53/TP53, SREBF1, SREBF2 and PPARGC1A. Acts as a key regulator of glucose homeostasis in liver by phosphorylating CRTC2/TORC2, leading to CRTC2/TORC2 sequestration in the cytoplasm. In response to stress, phosphorylates 'Ser-36' of histone H2B (H2BS36ph), leading to promote transcription. Acts as a key regulator of cell growth and proliferation by phosphorylating TSC2, RPTOR and ATG1: in response to nutrient limitation, negatively regulates the mTORC1 complex by phosphorylating RPTOR component of the mTORC1 complex and by phosphorylating and activating TSC2. In response to nutrient limitation, promotes autophagy by phosphorylating and activating ULK1. AMPK also acts as a regulator of circadian rhythm by mediating phosphorylation of CRY1, leading to destabilize it. May regulate the Wnt signaling pathway by phosphorylating CTNNB1, leading to stabilize it. Also has tau-protein kinase activity: in response to amyloid beta A4 protein (APP) exposure, activated by CAMKK2, leading to phosphorylation of MAPT/TAU; however the relevance of such data remains unclear in vivo. Also phosphorylates CFTR, EEF2K, KLC1, NOS3 and SLC12A1.
  • Alternative names
    • 5 AMP activated protein kinase catalytic subunit alpha 1
    • ACACA kinase
    • Acetyl CoA carboxylase kinase
    • AMPK
    • AMPK subunit alpha 1
    • AMPKa1
    • HMGCR kinase
    • Hydroxymethylglutaryl CoA reductase kinase
    • Tau protein kinase PRKAA1
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab181422 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
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Background-subtracted data values (mean +/- SD) are graphed. 

  • Background-subtracted data values (mean +/- SD) are graphed.

  • Background-subtracted data values (mean +/- SD) are graphed. 

  • Background-subtracted data values (mean +/- SD) are graphed. 

  • Interpolated values of AMPK-alpha 1 are plotted for the indicated cell lines at 100 µg/mL lysate loading. Corresponding Western Blot bands at 40 ug per lane loading are shown for the capture (A) and detector (B) antibodies. Hek293T cells show significantly higher levels of AMPK-alpha 1 both by western blot and by SimpleStep ELISA

  • Endogenous levels of AMPK-alpha 1 were measured from various cell extracts on western blot using the capture and detector antibodies provided in the kit. Cells were extracted using the protocol outlined in Section 11 and supplemented with SDS sample buffer prior to loading. Samples were loaded as follows: (1) 40 µg Hek293T, (2) 40 µg HeLa, (3) 40 µg MCF7, and (4) 40 µg HepG2. Samples 2-5 were blotted with the detector antibody, and samples 6-9 were blotted with the capture antibody. Blots were performed under reducing conditions. Membranes were blocked with 5% Milk in TBS + 0.1% Tween-20 (TBST) for 1 hour at room temperature. Primary antibodies were incubated in 1% Milk in TBST overnight. Secondary antibodies (goat anti-rabbit HRP for detector and goat anti-mouse HRP for capture) were incubated in 1X Blocking Buffer (ab126587) for 2 hours at room temperature.

Protocols

References

This product has been referenced in:
  • Müller-Durovic B  et al. Killer Cell Lectin-like Receptor G1 Inhibits NK Cell Function through Activation of Adenosine 5'-Monophosphate-Activated Protein Kinase. J Immunol 197:2891-9 (2016). Read more (PubMed: 27566818) »

See 1 Publication for this product

Customer reviews and Q&As

Abreviews
Product worked well in human plasma (1:200 dilution), but didn't work on human CSF (even non-diluted or lysed CSF). We are trying concentrated CSF using a separate kit. We'll update with those results (either positive or negative results).
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Verified customer

Submitted Jun 23 2017

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