In Vitro Sumoylation
In Vitro Sumoylation - Related Videos
06:00
SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting
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2025
This video demonstrates the in vitro method for SUMOylation of substrate proteins using a sequential enzyme cascade. Further, the SUMOylated status of the protein is identified using the electrophoresis and immunoblotting technique.
09:40
In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells
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Cited by 2 •
2017
Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.
09:45
In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
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Cited by 11 •
2018
Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.
08:25
Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure
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Cited by 21 •
2020
This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.
08:03
An In Vitro Organ Culture Model of the Murine Intervertebral Disc
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Cited by 22 •
2017
Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.
FAQs
ATP first enables the E1 enzyme to activate SUMO, establishing the energy-dependent starting point of the reaction. Ubc9 then transfers the activated modifier toward a substrate lysine, where covalent attachment occurs. This ordered sequence allows researchers to examine individual stages of SUMO conjugation rather than observing only the final modification in a cell.
Ubc9 functions as the transfer enzyme that directs SUMO toward a lysine residue on the substrate. Its position between E1-mediated activation and substrate modification makes it central to the conjugation pathway. Studying this step helps researchers determine whether a target can receive SUMO and distinguish substrate compatibility from effects caused by other components of the reaction.
An E3 ligase can improve recognition of a particular substrate and increase the efficiency of its modification. Comparing reactions with and without the ligase can therefore reveal whether substrate recognition depends on this additional factor. This comparison is useful for examining specificity and for evaluating how regulatory components influence the extent of SUMO attachment.
A reconstituted reaction brings together SUMO, ATP, the E1 activating enzyme, Ubc9, and a protein substrate containing a candidate lysine modification site. An E3 ligase may be added when enhanced recognition or efficiency is being investigated. Combining these defined components lets researchers attribute the observed modification to the reconstructed SUMO-conjugation system.
Different amounts of attached SUMO can indicate changes in substrate recognition, conjugation efficiency, or the contribution of an E3 ligase. Because the reaction is cell-free and controlled, researchers can test these factors systematically and compare substrate behavior under defined conditions. The resulting patterns help evaluate specificity and clarify which parts of the cascade influence modification.
The approach is valuable when researchers need to test a suspected SUMO substrate, examine regulatory effects, or validate an observation made in cells. It can support mechanistic studies connected to transcription, DNA repair, and protein trafficking by isolating the modification step. Results can reveal whether SUMO attachment is consistent with changes in protein activity, stability, localization, or interactions.