Capture more ubiquitin-modified proteins!

The Pierce Ubiquitin Enrichment Kit facilitates the isolation of polyubiquitin protein conjugates from cultured cells and tissue samples. The enriched fraction can be subsequently analyzed to determine the amount of general ubiquitin conjugates present or to identify a specific protein of interest by Western blotting. The assay protocol is fast and straightforward (see summary below), allowing for isolation of polyubiquitinated proteins and the fractionation of monoubiquitinated species in the resin flow-through. The kit provides exceptional performance in enriching an epoxomicin-treated HeLa lysate for polyubiquitinated proteins (Figure 1). The Ubiquitin Enrichment Kit outperforms similar resins from other suppliers and provides a clean and specific preparation of proteins when compared to a control resin.

Figure 1. The Thermo Scientific Pierce Ubiquitin Enrichment Kit recovers more ubiquitin-modified proteins than any other method. Epoxomicin-treated HeLa cell lysates (EHeLa, 150 µg) were enriched by the Pierce Kit (see procedure summary below), a competitor kit and an antibody-based methods. After elution, all samples were normalized to the initial load (EHeLa load). Right-most lanes (GSH resin) are a negative control to show that the resin does not bind proteins nonspecifically.

Procedure Summary

1. Lyse cells or tissue (150 mg, 150 ml).
2. Add 150 ml of sample dilution buffer and 20 ml of ubiquitin affinity resin.
3. Incubate at 4°C for 2 hours to overnight.
4. Centrifuge spin column at 5,000 x g for 15 seconds. (Save flow-through for later analysis.)
5. Wash resin 3 times with wash buffer. (Save wash flow-throughs for later analysis.)
6. Elute ubiquitinated protein sample with SDS-PAGE loading buffer or IEF buffer.
7. Perform Western blot analysis using anti-ubiquitin antiserum (included in kit) or antibody specific for your protein of interest (you supply).

The ubiquitin proteasome pathway is the principal non-lysosomal pathway that controls the proteolysis of proteins. This pathway is significantly involved in a variety of cellular processes, including DNA repair, transcriptional regulation, signal transduction, cell metabolism and morphogenesis. The ubiquitination process is initiated when ubiquitin, an 8 kDa polypeptide consisting of 76 amino acids, is appended to cellular proteins via the C-terminal glycine of the ubiquitin molecule. Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur. Polyubiquitinated proteins are then recognized by the 26S proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin. Differences in total ubiquitination or the ubiquitination of specific proteins affect numerous pathological conditions, including malignancies, certain genetic diseases and neurodegenerative diseases.¹

• Fast — less than 45 minutes hands-on time
• Complete — includes all reagents needed for ubiquitin-modified protein enrichment from cultured cells and tissue samples, including spin columns and ubiquitin antibody
• Flexible — sample incubation from 2 hours to overnight allows assay to be completed in several hours or in less than 30 minutes after overnight incubation
• Robust — compatible with all Pierce cell lysis reagents and standard RIPA formulations
• Multiple-sample format — easily processes 1-15 samples concurrently

Related Products

Phosphoprotein Enrichment Kit
Phosphopeptide Isolation Kit
Glycoprotein Isolation Kits

Reference

1. Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17(24), 7151-60.