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.
If you suspect that your protein may be sumoylated then the common first step in proving this is a standard co-IP of both SUMO and the target protein and we have a range of SUMO Affimer reagents waiting for you to use in your own pulldowns.
Unfortunately one of the main drawbacks in studying protein sumoylation is that endogenous modification is very hard to identify. Handily the following tips tackle just this problem. The recommendations included here aim to increase your chances of success in identifying those pesky sumo-modified proteins.
- 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.
Non-denatured lysate
To inhibit SENPs, the enzymes that remove sumo groups, it is essential to include the isopeptidase inhibitor N-ethylmailemide (NEM) at a final concentration of 15-20mM in the extraction buffer. Cell lysis should then be achieved by drawing the cell suspension through a 2-gauge needle 5 times and incubating on ice for 15 minutes. The lysate is then collected following high speed centrifugation for 20 minutes at 4°C.
Denatured lysate
Cell pellets should be suspended in modified 2 x Lamelli buffer (150 mM Tris·HCl pH 7.2, 4% SDS, 20% glycerol and 20 mM NEM; The re-activation of isopeptidases after the removal of SDS is possible, therefore, the inclusion of NEM in the denaturating buffer is still recommended). The sample sonicate to reduce viscosity and boiled at 100°C for 10 mins. Lysates should then be collected after high speed centrifugation at RT for 15 minutes. The denaturing lysate should be cleared of SDS prior to IP using detergent removal spin columns,
(Thermo Scientific, 87778).
- 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.
Following 3 washes with lysis buffer elute the proteins using 50?l of elution buffer for 5 minutes at RT.
Different sumoylated isoforms and target proteins may finally be detected by western blot as bands above the molecular weight corresponding to the non-sumoylated isoform of the protein (unless the sumoylated isoform is the predominant isoform, which is usually not the case).
These recommendations on improving the identification of sumoylated proteins come from a paper currently in press in the journal of Analytical Biochemistry.