The identification of novel substrates for E3 enzymes is most efficiently achieved through the use of protein microarray technology. E3 Substrate ID™ employs a high-content microarray and LifeSensors’ ubiquitin pathway expertise to identify candidate substrates for any ubiquitin E3 ligase. The service includes the following steps:[1] Determination of the optimal E2 for your E3 across twenty human E2s using our E2 Profiling Kit (cat. no. UC102).[2] A protein microarray is then ubiquitylated using an optimal E2/E3 pair. A second control array is incubated with the same solution, but no E3.[3] Ubiquitylated substrates are identified using ubiquitin detection (antibody or TUBEs). The arrays are scanned and differences between the signal intensity on the experimental array versus the control array are indicative of proteins that are ubiquitylated by the E3. We give you a report with a list of all proteins that show a statistically significant (p<0.05) difference between the two arrays. The protein list is also subjected to Gene Ontology analysis to sort by familial relationships. As we are not experts in the customer’s field, we leave it to them to filter the list(s) for proteins that may make biological sense.The service can be performed with one of several commercially available arrays or a custom array. LifeSensors can advise on the selection of the most appropriate array for the experiment. Screens have been performed successfully with protein arrays containing approximately 20,000 human proteins.
Array
Client-selected human protein microarray
Client material required
E3 of interest provided by client or selected from our catalog (roughly 150 µL of a 100 nM solution)
Deliverable
Raw data & statistical analysis, gene ontology (GO) clustering of potential substrates,follow-up support and lead prioritization.
Turnaround
4-6 weeks
Contact our scientists for a free consultation. Find out how LifeSensors’ Ubiquitin Protein Microarray Services can provide answers to your ubiquitome-related questions.
