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|7955S||1 Kit (Reagents for 4 x 96 well plates)||$459.00.0|
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PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955
Gallery: PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955
- Figure 1. The relationship between the protein concentration of lysates from untreated and H2O2-treated C2C12 cells and the absorbance at 450 nm using the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955 is shown.
ELISA Antibody Pair
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Wash Buffer: 1X PBS/0.05% Tween® 20, (20X PBST #9809).
- Blocking Buffer: 1X PBS/0.05% Tween® 20, 1% BSA.
1X Cell Lysis Buffer: PathScan® Sandwich ELISA Lysis Buffer (#7018) 1X: This buffer is ready to use as is. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
- Bovine Serum Albumin (BSA): (#9998).
- TMB Substrate: (#7004).
STOP Solution: (#7002)
NOTE: Reagents should be made fresh daily.
B. Preparing Cell Lysates
For adherent cells.
- Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
- Remove media and rinse cells once with ice-cold 1X PBS.
- Remove PBS and add 0.5 ml to 1 ml ice-cold PathScan® Sandwich ELISA Lysis Buffer (1X) #7018 plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 2 min.
- Collect cell lysate in a clean tube.
- 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 (~1,200 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 (~1,200 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 on ice 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. Coating Procedure
- Rinse microplate with 200 μl of dH2O, discard liquid. Blot on paper towel to make sure wells are dry.
- Dilute capture antibody 1:100 in 1X PBS. For a single 96 well plate, add 100 μl of capture antibody stock to 9.9 ml 1X PBS. Mix well and add 100 μl/well. Cover plate and incubate overnight at 4°C (17–20 hr).
- After overnight coating, gently uncover plate and wash wells:
- Discard plate contents into a receptacle.
- Wash four times with wash buffer, 200 μl each time per well. For each wash, strike plates on fresh paper 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.
- Block plates. Add 150 μl of blocking buffer/well, cover plate, and incubate at 37°C for 2 hr.
- After blocking, wash plate (Section C, Step 3). Plate is ready to use.
D. Test Procedure
- Lysates can be used undiluted or diluted in blocking buffer. 100 μl of lysate is added per well. Cover plate and incubate at 37°C for 2 hr.
- Wash plate (Section C, Step 3).
- Dilute detection antibody 1:100 in blocking buffer. For a single 96 well plate, add 100 μl of detection antibody Stock to 9.9 ml of blocking buffer. Mix well and add 100 μl/well. Cover plate and incubate at 37°C for 1 hr.
- Wash plate (Section C, Step 3).
- Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in blocking buffer. For a single 96 well plate, add 10 μl of secondary antibody stock to 9.99 ml of blocking buffer. Mix well and add 100 μl/well. Cover and incubate at 37°C for 30 min.
- Wash plate (Section C, Step 3).
- Add 100 μl of TMB substrate per well. Cover and incubate at 37°C for 10 min.
- Add 100 μl of STOP solution per well. Shake gently for a few seconds.
- Read plate on a microplate reader at absorbance 450 nm.
posted January 2008
revised January 2016
|Product Includes||Volume||Cap Color|
|AMPKα Rabbit Capture Rabbit mAb (100X)||400 µl||Pink|
|Phospho-AMPKα (Thr172) Detection Mouse mAb (100X)||400 µl||Blue|
|Anti-mouse IgG, HRP-linked Antibody (1000X)||40 µl||Yellow|
CST's PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-AMPKα-(Thr172) Sandwich ELISA Kit #7959. Capture and Detection antibodies (100X stocks) and Anti-Mouse IgG, HRP-linked Antibody (1000X stock) are supplied. Sufficient reagents are provided for 4 x 96 well ELISAs. The AMPKα Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a Phospho-AMPKα (Thr172) Mouse Detection Antibody and Anti-Mouse IgG, HRP-linked Antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-AMPKα (Thr172) protein.
Antibodies in kit are custom formulations specific to kit.
For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity: Human, Mouse
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 the activation of AMPK enzyme, 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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc.