Signaling Pathway Assays
AMPK Phosphorylation Assay Kit (Fluorometric) (BA0075)
- SKU:
- BA0075
- Product Type:
- Assay
- Detection Method:
- Fluorometric
- Instrument:
- Microplate Reader
- Sample Type:
- Cell, Tissue
- Research Area:
- Plant & Environmental Stress
Description
AMPK Phosphorylation Assay Kit (Fluorometric) (BA0075) (BA0075)
The AMPK Phosphorylation Assay Kit (Fluorometric) (SKU: BA0075) is a cell-based ELISA that measures phosphorylated AMPK in whole cells and normalises the signal to total protein content. The 5'-AMP-activated protein kinase (AMPK) is a key sensor of intracellular energy balance, activated by an increase in the AMP/ATP ratio caused by factors such as muscle contraction, starvation or hypoxia, and it is activated by phosphorylation on threonine-172 within its catalytic domain. The antibody recognises both alpha-subunits and can be used for cells from all tissues. This simple and efficient assay eliminates the need for cell lysate preparation: cells grown in 96-well plates are fixed and permeabilised in the wells, phosphorylated AMPK is measured using a fluorescent ELISA, and total protein is then measured in each well. It can be used to study AMPK regulation in both short-term and long-term assays.
| Product Name: | AMPK Phosphorylation Assay Kit (Fluorometric) (BA0075) |
| SKU: | BA0075 |
| Detection Method: | Fluorometric cell-based ELISA |
| Detection Range: | Can measure pAMPK modulation in as little as 500 cells per well |
| Sample Type: | Cultured cells |
| Species Reactivity: | Human, mouse and rat |
| Assay Time: | Approximately 6.5 hours total (about 2.5 hours hands-on) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Fluorimetric cell-based ELISA for AMPK phosphorylation status. Phosphorylated AMPK is detected with a fluorescent ELISA (excitation/emission 530/585 nm) and normalised to total protein (excitation/emission 360/450 nm) in the same well.
- Sensitive: can measure pAMPK modulation in as little as 500 cells per well
- New and improved with total assay time reduced to 6.5 hours
- Simple and convenient with cells cultured directly in 96-well plates and no cell lysis required
- Accurate and high-throughput, normalising phosphorylation to total cellular protein
- Quantitative fluorescent immunoenzymatic assay of AMPK phosphorylation status in cultured cells
- Evaluation of direct and indirect modulation of AMPK phosphorylation
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Preparation. Prepare 1x Wash Buffer by diluting Stock Wash Buffer 40-fold with distilled water and reserve 6 mL for the detection step. Assay samples in triplicate or higher and include a Protein Blank (no cells) and a Sample Blank (cells with only Ab2). |
| 2 | Culture and treat cells. Seed 100 uL of 1-3 x 10^4 adherent cells (or 4-10 x 10^4 suspension cells) per well of a black 96-well culture plate, add media-only wells for the Protein Blank and incubate overnight at 37C. Treat cells as desired, then fix with formaldehyde, wash, quench with hydrogen peroxide, wash again and block for 1 hour. |
| 3 | Add primary antibody. Prepare Ab1 mixture at 1:625 in Blocking Buffer, add 50 uL to sample wells (Blocking Buffer only to Sample Blanks), incubate 90 minutes at room temperature or overnight at 2-8C and wash three times. |
| 4 | Add secondary antibody. Prepare Ab2 mixture at 1:625 in Blocking Buffer, add 50 uL to all wells, incubate 90 minutes at room temperature and wash five times. |
| 5 | Detection. Prepare HRP substrate by mixing 60 uL Dye Reagent with 6 mL 1x Wash Buffer and 6 uL 3% hydrogen peroxide, add 50 uL to each well and incubate 30 minutes in the dark. Add 50 uL Protein Stain and incubate a further 5 minutes in the dark. Read at excitation/emission 530/585 nm for phosphorylated AMPK and 360/450 nm for total protein. |
Calculate mean pAMPK fluorescence (530/585 nm) for Sample Blank and Sample wells, and mean protein fluorescence (360/450 nm) for the Protein Blank and Sample wells. Subtract blanks to obtain the change in fluorescence for pAMPK and for total protein. Normalised pAMPK = (change in pAMPK / change in protein) divided by the same ratio for the control reference (e.g. time zero or untreated wells).
| Component | Quantity | Storage |
| Stock Wash Buffer | 25 mL | -20C |
| Blocking Buffer | 25 mL | -20C |
| Protein Stain | 6 mL | -20C |
| Dye Reagent | 120 uL | -20C |
| Ab1 | 10 uL | -20C |
| Ab2 (gR-HRP) | 10 uL | -20C |
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