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AMPKalpha (Phospho-Thr172) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00001
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Fluorometric
€599
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Description

Technical Manual

AMPKalpha (Phospho-Thr172) Cell-Based ELISA Kit

The AMPKalpha (Phospho-Thr172) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor AMPKalpha phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated AMPKalpha in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on AMPKalpha phosphorylation.

How does our AMPKalpha (Phospho-Thr172) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of AMPKalpha (Phospho-Thr172) concentration is achieved by an indirect ELISA format. In essence, AMPKalpha (Phospho-Thr172) is captured by AMPKalpha (Phospho-Thr172)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values.
2. An antibody against the nonphosphorylated counterpart of AMPKalpha (Phospho-Thr172) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated AMPKalpha can be used to normalize the RFU value for phosphorylated AMPKalpha.

AMPKalpha (Phospho-Thr172) Fluorometric Cell-Based ELISA Kit -Information

Product Name:AMPKalpha (Phospho-Thr172) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00001
Description:The AMPKalpha (Phospho-Thr172) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor AMPK (Phospho-Thr172) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated AMPK (Phospho-Thr172) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on AAPK1/AAPK2 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 5562/5563, UniProt ID: Q13131/P54646, OMIM #: 602739/600497, Unigene #: Hs.43322/Hs.437039
Format:Two 96-Well Plates
NCBI Gene Symbol:AAPK1/AAPK2
Sub Type:Phospho
Target Name:Phospho-AMPK? (Thr172)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components

Quantity

96-Well Black Cell CultureClear-Bottom Microplate2 plates
10X TBS24 ml
Quenching Buffer24 ml
Blocking Buffer50 ml
15X Wash Buffer50 ml
Primary Antibody Diluent12 ml
100x Anti-Phospho Target Antibody 60 µl
100x Anti-Target Antibody60 µl
Anti-GAPDH Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
  • Micropipettes capable of measuring volumes from 1 µl to 1 ml
  • Deionized or sterile water (ddH2O)
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for 3 minutes. The plate can be stored at 4°C for a week.
7.Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 3 minutes each time.
9.Dispense 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
11.Add 50 µl of Primary Antibody Mixture P to corresponding wells for AMPKalpha (Phospho-Thr172) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total AMPKalpha detection. Cover the plate with parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
13.Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

AMPKalpha (Phospho-Thr172) - Protein Information

UniProt Protein Function: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/ULK1: 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 ATG1/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.
NCBI Summary:The protein encoded by this gene belongs to the ser/thr protein kinase family. It is the catalytic subunit of the 5'-prime-AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensor conserved in all eukaryotic cells. The kinase activity of AMPK is activated by the stimuli that increase the cellular AMP/ATP ratio. AMPK regulates the activities of a number of key metabolic enzymes through phosphorylation. It protects cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. Alternatively spliced transcript variants encoding distinct isoforms have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:Q13131
NCBI GenInfo Identifier:254763436
NCBI Gene ID:5562
NCBI Accession:Q13131.4
UniProt Secondary Accession:Q13131,O00286, Q5D0E1, Q86VS1, Q9UNQ4, A8MTQ6, B2R7E1
UniProt Related Accession:Q13131
Molecular Weight:65,523 Da
NCBI Full Name:5'-AMP-activated protein kinase catalytic subunit alpha-1
NCBI Synonym Full Names:protein kinase AMP-activated catalytic subunit alpha 1
NCBI Official Symbol:PRKAA1  
NCBI Official Synonym Symbols:AMPK; AMPKa1  
NCBI Protein Information:5'-AMP-activated protein kinase catalytic subunit alpha-1
UniProt Protein Name:5'-AMP-activated protein kinase catalytic subunit alpha-1
UniProt Synonym Protein Names:Acetyl-CoA carboxylase kinase (EC:2.7.11.27
UniProt Gene Name:PRKAA1  

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