Sumoylation of proteins has been associated with a whole host of cellular processes; transcription, replication, DNA repair, RNA metabolism, translation and transport and consequently interest in this modification is growing. Sumoylation of proteins usually occurs through binding of this moiety to lysine residues within the target protein or through non-covalent interactions. Four Sumo paralogues have been identified, SUMO1, 2, 3 (which are often referred to as 2/3 as they share 95% sequence homology) and 4. SUMO4 expression is limited to liver, kidney and lymph nodes while the other three are widely expressed in different tissues.
- Only a small fraction of intracellular protein is sumoylated at any one time. To increase the likelihood of successful IP a highly concentrated protein lysate of 1×107– x108 cells in 600?l buffer percondition/control is recommended as a starting material. If this leads to a high background then further experiments can be performed to reduce this by reducing the protein concentration and/or increasing washes.
- Though a denaturing lysis buffer with high concentrations of SDS will inactivate the enzymes that remove sumo moieties from proteins preventing the loss of signal, they may also eliminate non-covalent interactions between sumo and target proteins leaving only those proteins covalently modified as sumoylated. This high concentration of SDS is usually removed or diluted significantly to allow sumo to bind during the IP step and its removal allows the non-covalent interactions to reform. As denaturing buffers can help with the extraction of difficult proteins it is reassuring to know that no difference was observed in the identification of sumoylated proteins extracted with denaturing or non-denaturing detergents.
- Pre-clearing of the cell lysate by incubating the lysates with beads to remove non-specific binding is recommended. Screw cap spin columns from Thermo Scientific (69705) are good to ensure successful washing without bead loss. Remember to keep an aliquot of pre-IP whole cell lysate aside for western blot analysis later! The IP should then follow a general procedure.
To be sure to pulldown all sumoylated protein in the IP the use of very high concentrations of SUMO affinity reagent are advised. The IP is incubated overnight with anti-SUMO beads with gentle agitation, using IgG-cross linked beads as a control.