The PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-AMPKα (Thr172) protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using smaller samples. An AMPKα Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, AMPKα (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-AMPKα (Thr172) Mouse Detection Antibody is added to detect the captured phospho-AMPKα (Thr172) protein. HRP-linked, anti-mouse antibody is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of phospho-AMPKα (Thr172) protein.
*Antibodies in this kit are custom formulations specific to kit.
NOTE: Refer to product-specific datasheets for assay incubation temperature. This chemiluminescent ELISA is offered in low volume microplates. Only 50 μl of samples or reagents are required in each microwell.
A. Solutions and Reagents
NOTE: Prepare solutions with purified water.
Microwell strips: Bring all to room temperature before use.
Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 0.5 ml of Detection Antibody Diluent (green solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 0.5 ml volume of reconstituted Detection Antibody to 5.0 ml of Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.
HRP-Linked Antibody*: Supplied lyophilized as a red colored cake or powder. Add 0.5 ml of HRP Diluent (red solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 0.5 ml volume of reconstituted HRP-Linked Antibody to 5.0 ml of HRP Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C.
Detection Antibody Diluent: Green colored diluent for reconstitution and dilution of the detection antibody (5.5 ml provided).
HRP Diluent: Red colored diluent for reconstitution and dilution of the HRP‑Linked Antibody (5.5 ml provided).
Sample Diluent: Blue colored diluent for dilution of cell lysates.
1X Wash Buffer: Prepare by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in purified water.
Centrifuge for 10 min (14,000 x g) at 4°C and transfer the supernatant to a new tube. Store supernatant at -80°C in single-use aliquots.
For suspension cells
Remove media by low speed centrifugation (~1200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
Collect cells by low speed centrifugation (~1200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X Cell Lysis Buffer plus 1 mM PMSF.
Resuspend the cell pellet and incubate the tube for 2 min.
Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.
C. Test Procedure
After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed and stored at 4°C immediately.
Cell lysates can be undiluted or diluted with Sample Diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets for each kit provide a sensitivity curve that serves as a reference for selection of an appropriate starting lysate concentration. The sensitivity curve shows typical kit assay results across a range of lysate concentration points.
Add 50 μl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at room temperature. Alternatively, the plate can be incubated overnight at 4°C.
Gently remove the tape and wash wells:
Discard plate contents into a receptacle.
Wash 4 times with 1X Wash Buffer, 150 μl each time for each well.
For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
Clean the underside of all wells with a lint-free tissue.
Add 50 μl of reconstituted Detection Antibody (green color) to each well (refer to Section A, Step 2). Seal with tape and incubate the plate at room temperature for 1 hr.
Repeat wash procedure (Section C, Step 4).
Add 50 μl of reconstituted HRP-linked secondary antibody (red color) to each well (refer to Section A, Step 3). Seal with tape and incubate the plate at room temperature for 30 min.
Repeat wash procedure (Section C, Step 4).
Prepare Detection Reagent Working Solution by mixing equal parts Luminol/Enhancer Solution and Stable Peroxide Buffer.
Add 50 μl of the Detection Reagent Working Solution to each well.
Use a plate-based luminometer to measure Relative Light Units (RLU) at 425 nm within 1–10 min following addition of the substrate. Optimal signal intensity is achieved when read within 10 min.
posted November 2013
revised January 2016
Protocol Id: 203
Specificity / Sensitivity
PathScan® Phospho-AMPKα (Thr172) Chemiluminescent Sandwich ELISA Kit detects endogenous levels of phospho-AMPKα (Thr172), as shown in figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation (3-5). AMPKα is also phosphorylated at Thr258 and Ser485 (for α1; Ser491 for α2). The upstream kinase and the biological significance of these phosphorylation events have yet to be elucidated (6). The β1 subunit is post-translationally modified by myristoylation and multi-site phosphorylation including Ser24/25, Ser96, Ser101, Ser108, and Ser182 (6,7). Phosphorylation at Ser108 of the β1 subunit seems to be required for AMPK activation, while phosphorylation at Ser24/25 and Ser182 affects AMPK localization (7). Several mutations in AMPKγ subunits have been identified, most of which are located in the putative AMP/ATP binding sites (CBS or Bateman domains). Mutations at these sites lead to reduction of AMPK activity and cause glycogen accumulation in heart or skeletal muscle (1,2). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (1).