-->

Phospho-ULK1 (Ser757/Ser758) cellular kit HTRF®

The phospho-ULK1 (Ser757 mouse / Ser758 human) kit enables the cell-based detection of phosphorylated ULK1, one of the readouts for the AKT/mTOR pathway.
See more
  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The phospho-ULK1 (Ser757 mouse / Ser758 human) kit enables the cell-based detection of phosphorylated ULK1, one of the readouts for the AKT/mTOR pathway.
-

Overview

This Phospho-ULK1 cell-based assay is designed to monitor the modulation of ULK1 phosphorylated either on Serine 757 in mouse or Serine 758 in human. It is hallmark of autophagy, which is the processs of cellular eating.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SPECIFICITY

Phospho-ULK1 (Ser757mouse/Ser758human) assay principle

The Phospho-ULK1 (Ser757mouse/Ser758human) assay measures ULK1 when phosphorylated at Ser757mouse/Ser758human. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-ULK1 (Ser757mouse/Ser758human) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-ULK1 (Ser757mouse/Ser758human) assay principle

Phospho-ULK1 (Ser757mouse/Ser758human) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-ULK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-ULK1 (Ser757mouse/Ser758human) 2-plate assay protocol

Phospho-ULK1 (Ser757mouse/Ser758human) 1-plate assay protocol

Detection of Phosphorylated ULK1with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-ULK1 (Ser757mouse/Ser758human) 1-plate protocol

HTRF assay vs Western Blot using phospho-ULK1 cellular assay

HEK293 human embryonic kidney cells were cultured for 2 days at until 80% confluency was reached. The cells were then lysed with supplemented lysis buffer and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and transferred into a 384-well low volume white microplate before finally adding HTRF phospho-ULK1 kit reagents. A side by side comparison of Western Blot and HTRF demonstrated the HTRF assay is 4-fold more sensitive than the Western Blot.
HTRF assay vs Western Blot using phospho-ULK1 cellular assay

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..
MCF-7 cells + Torin1, Dose-response experiment

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
HEK293 cells + Wortmannin, Dose-response experiment

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.
MCF-7 cells + Insulin, Cell density experiment

Function and regulation of the ULK1 kinase complex

ULK1 kinase complex signaling pathway
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.

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert ULK1 P-S758 Kit / 64ULKPEG-64ULKPEH

64ULKPEG-64ULKPEH - Product Insert

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader

Latest news