Ubiquitination Assay

PROTACs mediate the degradation of target protein by hijacking the activity of E3 ubiquitin ligases for protein ubiquitination and subsequent degradation by the 26S proteasome. Therefore, detection of protein ubiquitination is a key step in determining the success of PROTAC.

Profacgen provides multiple technical platforms ubiquitination analysis:

Immunoprecipitate (IP) and Western blot

This is the simplest and more direct technique to determinate target protein ubiquitination. After immunoprecipitation of target protein from lysate, Western blot is performed with specific antibodies against ubiquitination molecule. This technique has an advantage in measuring the ubiquitination of endogenous proteins; and inhibitors can improve the signal in case of proteins degraded.

Enzyme-linked Immunosorbent Assay (ELISA)

The Dissociation Enhanced Lanthanide Fluoroimmunoassay (DELFIA) is a quantitative ELISA based-methodology to detect protein ubiquitination in cells, and allows more samples and experimental conditions to be compared in parallel. This technology is based on sandwich recognition of a target protein by a capture antibody and a detection antibody. After immobilization of the capture antibody on a solid surface, the analyte (appropriate cellular lysate) is incubated on the plate. Ubiquitinated proteins will produce a specific signal when incubation with the detection antibody.

Fluorescence polarization (FP)

This method uses the fluorescent molecule to track the progression of E1, E2, and E3 enzymes along with the FP changes caused by molecular weight. In FP assay, ubiquitination activities are observed in a parallel manner and consumes only fluorescent molecules. This assay is useful in HTS for small molecule modulators and alternative to mechanistic work.

Mass Spectrometry (MS)

Recently MS has become an indispensable tool for proteome analyses like posttranslational modifications (PTMs) of proteins (e.g., phosphorylation, methylation, acetylation, and ubiquitination) which can modulate protein activity. In a MS assay, after tryptic digestion of ubiquitinated proteins, peptides conjugated to the epsilon amino group of lysine can be used to track back the original modification site. Efficient immunopurification of peptides combined with sensitive detection by MS has resulted in a huge increase in the number of ubiquitination identify.

Bioluminescence Resonance Energy Transfer (BRET)

BRET is a proximity-based methodology that detects ubiquitination in intact cells and in real time. In a BRET system, the occurrence of a resonance energy transfers (protein interactions) between a luminescent donor and a fluorescent acceptor protein. This is a system for monitoring constitutive and regulated protein interactions relying on the strict molecular proximity between the donor and acceptor.

The workflow of our services:

1. Identify demand and project design.

2. Sample submission and quality control.

3. Measurement and progress communication.

4. Data analysis and report delivery.

As leading research institutions, Profacgen offers one-step services with superior performance and short turnaround time. Please contact us for more information.

References:

1. Sigismund S, Polo S. (2016). Strategies to Detect Endogenous Ubiquitination of a Target Mammalian Protein. Proteostasis, 143–151. doi:10.1007/978-1-4939-3756-1_6
2. Franklin TG, Pruneda JN. (2019). A High-Throughput Assay for Monitoring Ubiquitination in Real Time. Frontiers in Chemistry, 7. doi:10.3389/fchem.2019.00816
3. Bezstarosti K, van der WalL, Demmers JAA. (2020). Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry. Journal of Visualized Experiments, (157). doi:10.3791/59079
4. Nagi K, Shenoy SK. (2019). Detection of β-Arrestin-Mediated G Protein-Coupled Receptor Ubiquitination Using BRET. Applied Mathematical Sciences, 93–104. doi:10.1007/978-1-4939-9158-7_6