LifeSensors’ TUBE (Tandem Ubiquitin Binding Entity) technologies are industry-leading tools to detect and enrich/purify poly-ubiquitylated proteins from cell lines, tissues, and organs. LifeSensors has two commercially available pan-TUBEs (TUBE1 and TUBE2). Both TUBE1 and TUBE2 come in various flavors such that they can be used for far-Western detection (FLAG, Biotin), enrichment/purification (linked to Agarose and Magnetic beads), and in vivo imaging (TAMRA, FITC-attached) applications. For more information, please see our TUBEs page.
TUBE1 and TUBE2 are made from different ubiquitin-binding domains (UBDs). However, both of them have been shown to bind all the linkages of polyUb with single digit nM Kd. TUBE1 has been shown to preferentially bind K63-polyUb over K48-polyUb. TUBE2 binds both K48- and K63-polyUb with equal affinities.
LifeSensors have recently developed a new K48 TUBE HF (high-fidelity) that has comparable affinity (Kd of ~20 nM for tetra-ubiquitin) to their cognate poly-Ub as our existing K63 TUBE and M1 TUBE. The old K48TUBE binds tetra-ubiquitin with a Kd of ~200 nM.
5. Which TUBE is more efficient at capturing human proteins as opposed to rodents, plants or yeasts?
TUBEs bind to ubiquitin/polyubiquitin chains which are highly conserved across all eukaryotes. However, the poly-ubiquitin linkage type (K48, K63) and the relative abundance of target protein should be considered while making a selection of TUBEs for your experiments.
No, TUBEs have not been tested by LifeSensors for organisms other than mammalian and yeast ubiquitin/poly-ubiquitins. Since ubiquitin is highly conserved from yeast to human, theoretically TUBEs should work for plants and other organisms. We welcome any feedback from customers who have studied other organisms with our TUBEs.
The abundance of the target protein and the type of sample (cells, tissues, organs) should be considered to estimate the amount of cell/tissue extracts needed. As a starting point, we recommend to use 20 µL of agarose -TUBE beads or 100 µL magnetic-TUBE slurry per milligram of cell extract. The amount of cell/tissue extracts, volume of the eluate, and the volume of the agarose/magnetic beads should be optimized by the end user.
Elute poly-ubiquitylated proteins from agarose/magnetic-TUBEs using our proprietary elution buffer (LifeSensors Cat # UM411B). Alternatively, for gel-based separation and subsequent applications, the ubiquitylated proteins can be eluted using a standard loading dye for SDS-PAGE.
Buffers containing chaotropic agents, such as urea and guanidinium-HCl, can be used for eluting poly-ubiquitylated proteins for subsequent mass spectrometric analysis. Alternatively, poly-ubiquitylated proteins can be eluted using SDS-PAGE loading buffer, separated by SDS-PAGE and subjected to trypsin digestion prior to MS analysis.
Positive control: How do I know that TUBEs work?
The properties of TUBEs have been well characterized and published. Each TUBE lot is quality controlled for overall binding capacity and the pull-down efficiency of polyubiquitylated proteins using cell lysate.
Negative control: How do I know that TUBEs aren’t yielding an artifact?
We recommend to include a negative control in your pull-downs that contains the quenched agarose resin (LifeSensors cat # UM400).
It is possible that under certain conditions significant amounts of mono-ubiquitylated proteins can be enriched/purified using TUBEs. This is particularly true for more abundant mono-ubiquitylated proteins. However, under typical cellular conditions, the majority of the isolated ubiquitins are likely to be polyubiquitylated.
LifeSensors has successfully applied the TUBE technology to detect and quantify the ubiquitylation status of a tagged/endogenous protein (UbiTest, UbiQuant S), detect the ubiquitin linkages (linkage-selective UbiTest), and quantify the ubiquitylated target protein in a high throughput format (HT-UbiTest).
The UbiTest platform replaces traditional immunoblotting methods for measuring cellular ubiquitylation, enabling simple comparisons of substrate ubiquitylation levels between samples and determination of ubiquitin chain linkage types. UbiTest is based on TUBE technology for enriching polyubiquitylated proteins. By visualizing the ubiquitylated fraction both with and without DUB digestion of polyubiquitin chains, UbiTest streamlines the confirmation of ubiquitylation status. For more information about UbiTest, please also visit our UbiTest product pages, where you can purchase off-the-shelf assay kits for performing UbiTest in your own lab.
LifeSensors has also developed High-Throughput (HT) UbiTest which is a customized medium-throughput assay for absolute quantification of ubiquitylated substrate levels in cells that combines UbiTest principles and TR-FRET technology. Please contact us if you like to develop similar assays for your target protein.
LifeSensors’ UbiQuant S ELISA and AlphaLISA assays are industry-leading platforms for measuring ubiquitylation of a target protein in cells. These assays utilized TUBEs (Tandem Ubiquitin Binding Entities) to detect ubiquitylated substrates, and are individually optimized for a specific substrate protein.
Yes, LifeSensors offers services to customize and develop assays required for your target protein. Additionally, LifeSensors has the expertise to determine compound activity in a cellular context. Our assays for monitoring cellular ubiquitylation will help you quickly demonstrate the downstream effects of your E3 ligase or DUB inhibitor. Ranging in throughput from immunoblot to 384-well formats, our assay concepts can be readily adapted for a vast range of substrates on a custom basis. Thus, our technologies will enable the advancement of your compounds from in vitro lead compounds to in vivo and preclinical models. For more information, please contact us or visit our service pages.
Yes, LifeSensors offers TUBE-based proteomics services. LifeSensors have developed two proprietary technologies to help researchers accomplish their scientific goal – (1) identify cellular proteins that are ubiquitinated and characterize the ubiquitin-linkages on them (TUBE proteomics), and (2) qualitative and quantitative profiling of ubiquitination sites on proteins (UbiSight proteomics). For more information, please contact us or visit our service pages.