TheEpiQuik™ In Vivo Protein Sumoylation Assay Ultra Kitis a complete set of optimized reagents designed for measuring in vivo protein SUMOylation from multiple mammalian cells/tissues including human, mouse, and rat.The kit has the following advantages:

• Fast procedure, which can be finished within 5 hours.
• Direct colorimetric assay without the need for affinity chromatography or Western blot.
• Flexible choice of antibody of interest allows for the detection of SUMOylation of multiple target proteins simultaneously.
• Use of optimized detection antibody eliminates the step for detection solution preparation, thereby increasing detection sensitivity and assay convenience.
• The positive control (SUMO protein) and negative control (un-SUMOylated non-immune IgG protein) allows protein SUMOylation to be quantified.
• Strip microplate format makes the assay flexible: manual or high throughput analysis.
• Reliable and consistent assay conditions.

Background InformationSUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. SUMO proteins are similar to ubiquitin. There are 3 confirmed SUMO isoforms in humans: SUMO-1, SUMO-2, and SUMO-3. SUMO-2/3 show a high degree of similarity to each other and are distinct from SUMO-1. SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. SUMOylation of target proteins in vivo has been shown to cause a number of different outcomes, including altered localization and binding partners. Detection of in vivo protein SUMOylation (SUMO conjugation) would provide useful information for understanding SUMO modification that emerges as an important control mechanism regulating the activity of many nuclear proteins.

Principle & ProcedureIn an assay with this kit, the antibodies specific to the targeted proteins are stably bound to the strip wells and the targeted proteins are captured by these antibodies. SUMOylation of the targeted proteins are detected by recognition of SUMO conjugated to these proteins with an anti-SUMO antibody. The ratio or intensity of the SUMOylation, which is proportional to the conjugated SUMO amount, can be quantified through the signal report-color development system by using a microplate reader at 450 nm wavelength.

Starting MaterialsInput material is nuclear extract. The amount of nuclear extracts for each assay can be 1 µg to 20 µg with an optimal range of 5 to 10 µg.

Fig. 1. Schematic Procedure for Using the EpiQuik™ In Vivo Protein Sumoylation Assay Ultra Kit (Colorimetric).

Fig. 2.Illustrated standard curve generated with SUMO protein control.