SUMOylation kit

Unopened kit should be stored at -80°C to ensure stability and activity. After opening, The SUMO antibody solutions (BML-PW8330 and BML-PW9465) can be stored at -20°C or -80°C. Other components should be stored at -80°C.

Uses: 1. SUMO-modification of specific proteins in vitro . Allow investigation of the effect SUMOylation has on enzyme function, stabilisation, protein:protein interactions and, hence, it’s role in regulation of cellular processes, such as the p53 tumour repressor and NF-κB pathways. 2. Demonstrate novel proteins are potential targets for SUMOylation under in vitro conditions. Starting point for examining the role SUMOylation of a protein might play in vivo . 3. Generate substrates for deSUMOylating enzymes, such as SENP1 (Prod. No. BML-UW9760) and SENP2 (Prod. No. BML-UW9765) . 4. Test proteins for SUMO E3 ligase activity: does it facilitate or enhance SUMOylation of specific target proteins, particularly under conditions/enzyme concentrations that more closely represent those in vivo. 5. Addition of known SUMO E3 ligase to facilitate/enhance target protein SUMOylation, particularly under conditions/enzyme concentrations that more closely represent those in vivo (e.g. RANBP2 [Prod. No. BML-UW9455], shown to be a ligase for SP100 SUMOylation) . 6. SUMOylation of proteins in cell lysates or crude fractions/preparations to facilitate investigation of their role/function in complex solutions. 7. Demonstrate SUMOylation of known proteins in specific lysates (confirm with target protein specific antibodies) . 8. Use of cell lysate or crude fractions/preparations as source of SUMO E3 ligases to facilitate SUMOylation of purified target proteins in the presence of SUMOylation kit components. Note: Protocol provided for application 1. Assay set-up can be readily modified for alternative applications by inclusion, omission or substitution of specific components.

20x SUMO Activating Enzyme Solution (SUMO E1) : SUMO activating enzyme E1 (human), (recombinant), (Prod. No. BML-UW9330), 20 μl. 20x SUMO Conjugating Enzyme Solution (SUMO E2) : Ubc9 (human), (recombinant) (untagged), (Prod. No. BML-UW9320), 20 μl. 20x SUMO Enzyme Solutions (SUMO-1, SUMO-2, SUMO-3) : SUMO-1 (human), (reccombinant) (His-tag), (Prod. No. BML-UW9195), 20 μl; SUMO-2 (human), (reccombinant) (His-tag), (Prod. No. BML-UW9205), 20 μl; SUMO-3 (human), (reccombinant) (His-tag), (Prod. No. BML-UW9215), 20 μl. 10x SUMOylation Buffer (Prod. No. BML-KW9890), 40 μl 20x Control RanGAP1 SUMOylation Target Protein Solution: RanGAP1 fragment (human), (recombinant) (GST-tag), (Prod. No. BML-UW9755), 20 μl. 20x Mg-ATP Solution (Prod. No. BML-EW9805), 25 μl SUMO Antibody Solutions: SUMO-1 (human) (NT), polyclonal antibody, (Prod. No. BML-PW8330), 25 μl; SUMO-2/3 (human) (NT), polyclonal antibody, (Prod. No. BML-PW9465), 25 μl.