ELISA Based Protein Ubiquitylation Measurement

Background

Protein ubiquitylation facilitates proteasomal and lysosomal degradations of misfolded proteins such as ∆F508 CFTR (Cystic Fibrosis Transmembrane conductance Regulator), resulting in the genetic disease, Cystic Fibrosis. In addition, protein ubiquitylation regulates selective autophagy, receptor endocytosis, and intracellular signal transductions. K48-linked poly-ubiquitylation and K63-linked poly-ubiquitylation facilitates proteasomal and lysosomal degradations, respectively. Therefore, quantitative analysis of protein ubiquitylation and specific poly-ubiquitin chain configurations of the protein-of-interest present as significant information in understanding the ubiquitylation-mediated processes.

Immunoblot detection of immunoprecipitated proteins is a commonly used technique to analyze protein ubiquitylation. This technique has an advantage in measuring the ubiquitylation of endogenous proteins; however, it is immensely demanding and time-consuming with relatively low sensitivity. In contrast, the ELISA-based ubiquitylation assay is simple and can easily quantitate the ubiquitylation levels of the protein-of-interest tagged with biotin when compared to immunoblot detection (Okiyoneda et al., 2018). The protein biotinylation can be achieved by introducing an HB (histidine-biotin) tag (Tagwerker et al., 2006) or AviTag^TM on the protein-of-interest. The biotinylated protein can be easily immobilized on streptavidin or NeutrAvidin-coated 96 well plates and denatured by urea to dissociate the interacting proteins. We prefer NeutrAvidin-coated 96 well plates in this protocol due to NeutrAvidin which has a higher affinity and specificity against the biotin-tag compared to streptavidin. The ubiquitin (Ub) chains conjugated to the protein-of-interest can be quantitated by ELISA using anti-Ub antibodies, including poly-ubiquitin chain specific antibodies. Few ubiquitin ELISA kits are commercially available (e.g., UbiQuant^TM quantitative ubiquitin ELISA from LifeSensors) and few ubiquitin-related ELISA methods are published as well (Conte et al., 2017). These published ELISAs use antibodies to immobilize ubiquitin or substrate proteins on multi-well plates. For some substrate proteins, the suitable antibody for the immobilization may be unavailable. In contrast, our method uses the interaction between Neutravidin and biotin in order to immobilize the biotinylated substrate proteins on multi-well plates. The interaction between Neutravidin and biotin exhibits the highest known affinity with high specificity. Moreover, it can withstand the presence of denaturing agents (e.g., 2 M urea), which dissociates the substrate-binding proteins, which may also be ubiquitinated. Thus, our method is advantageous over the other methods due to the unnecessary use of antibodies for the immobilization and specific detection of the substrate ubiquitination. The ELISA-based ubiquitylation assay potentially detects ubiquitylation of endogenous proteins genetically introduced with an HB tag or AviTag^TM by the CRISPR-Cas9 system. Through this protocol, we use the BHK cells stably expressing HBH (histidine-biotin-histidine) -∆F508 CFTR-3HA which were generated as previous work (Okiyoneda et al., 2018) while the ubiquitylation of ∆F508 CFTR tagged with an HBH tag can be measured by ELISA.