Phospho-ULK1 (Ser757 mouse / Ser758 human) Assay Kit

Phospho-ULK1 assay for monitoring the regulation of autophagy in cells

Autophagy is a cellular process of “self-eating”, defined as the lysosomal degradation of old or damaged intracellular components to generate nutrients and energy. The ULK1 kinase is the key initiator of the autophagic process in mammalian cells. The protein is activated by AMPK in response to nutrient starvation and cellular stresses, leading to autophagy up-regulation, cell survival and homeostasis. In presence of nutrients and growth factors, mTOR phosphorylates ULK1 on Ser758, which inactivates the kinase and leads to autophagy down-regulation. Excessive or deficient autophagy is associated with cancers, immune/infectious diseases, neurodegeneration and cardio-metabolic disorders.

This cell-based assay is designed to monitor the modulation of phosphorylated ULK1 (Serine 757 in mouse / Serine 758 in human) which is a hallmark of autophagy signaling.

Assay Principle

HTRF® - the homogeneous sandwich immunoassay

Cisbio Bioassay’s Phospho-ULK1 assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-ULK1 antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with ULK1, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assay can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho-ULK1 can be quantitatively detected using the HTRF phospho-ULK1 (Ser758) cellular assay kit reagents and most TR-FRET multimode plate readers

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred into a 384-well low volume white microplate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treatment and detection are all performed in a single plate. No washing steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling miniaturization while maintaining a high quality output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

Function and regulation of the ULK1 kinase complex

ULK1 is a Ser/Thr kinase which forms a complex with the Atg13 and FIP200 proteins. This complex is the most upstream component of the core autophagy machinery, and is therefore the key initiator of autophagy in mammalian cells.

ULK1 is regulated by the key nutrient/energy-sensitive kinases mTOR and AMPK, which are both able to phosphorylate ULK1 and directly regulate its kinase activity.

  • AMPK positively regulates autophagy: under nutrient starvation and cellular stresses, AMPK associates with and phosphorylates ULK1 on multiple sites, including Ser317 and Ser556. This leads to the activation of the complex and the initiation of the autophagic process. In these conditions, AMPK also inhibits mTOR.
  • mTOR plays a negative role in this process: under growth factor and nutrient signals, mTOR phosphorylates ULK1 on Ser758, which disrupts the interaction between ULK1 and AMPK and inhibits the autophagic process.  

Product Performance

1. HTRF assay compared to Western Blot using phospho-ULK1 cellular assay on human HEK293 cells

HEK293 human embryonic kidney cells were seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2, until 80% confluency was reached. The cells were then lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10 minute centrifugation.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-ULK1 detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.

Using HTRF® Phospho-ULK1, just 1,250 cells were sufficient for minimal signal detection, while 5,000 cells were needed for a Western Blot signal. The HTRF® assay is 4-fold more sensitive than the Western Blot.

2. Autophagy upregulation in MCF-7 human breast cancer cells using the mTOR inhibitor Torin 1

MCF-7 cells were plated at 100,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 5 hours with increasing concentrations of Torin 1, the medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-ULK1 (Ser758) detection reagents were added.

The HTRF signal was recorded after an overnight incubation.

3. Autophagy upregulation in HEK293 human embryonic kidney cells using the PI3K inhibitor Wortmannin

HEK293 cells were plated at 50,000 cells/well in a 96-well plate, and incubated for 24h at 37°C, 5% CO2. After treatment for 5 hours with increasing concentrations of Wortmannin, the medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-ULK1 (Ser758) detection reagents were added.

The HTRF signal was recorded after an overnight incubation.

4. AUTOPHAGY DOWNREGULATION IN MCF-7 HUMAN BREAST CANCER CELLS USING INSULIN

MCF-7 cells were plated at different cell densities in a 96-well plate and incubated for 24h at 37°C, 5% CO2. After a 3 hour serum-starvation step, cells were treated with 200 nM insulin for 30 minutes. The medium was then removed and cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-ULK1 (Ser758) detection reagents were added.

The HTRF signal was recorded after an overnight incubation.

 

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
ULK1 phospho-S758 kit - 500 tests64ULKPEG
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ULK1 phospho-S758 kit - 10,000 tests64ULKPEH
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Companion products

DescriptionCat. noProduct insertMSDS
ULK1 phospho-S758 kit - control lysate64ULKTDA
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