SUMOylated protein capture kit

Transcription

1 SUMOylated protein capture kit Cat no. A Capture and detect SUMOylated proteins High capacity, high specificity SUMO binding matrix Fast, convenient protein isolation using purification system provided Identify and analyse captured proteins by Western blotting or proteomic methods Contains sufficient SUMO binding matrix to perform up to 10 assays Please read this manual completely prior to using the product. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

2 Scientific background The post-translational modification of cellular proteins by Small Ubiquitin-like Modifier (SUMO) plays a role in a diverse range of biological processes including nuclear protein targeting, formation of sub-nuclear complexes, regulation of transcriptional activities, control of protein stability, chromosome segregation, recovery from checkpoint arrest, DNA damage response and meiosis. SUMOylation of substrate proteins takes place in a manner analogous to that of the ubiquitin conjugation system, utilising E1, E2 and E3 enzymes in an ATP dependent process and often requires a short sequence containing the consensus Ψ-K-X-D/E (where lysine is the modified amino acid, Ψ is a large hydrophobic residue and X is any amino acid residue) on the substrate protein for protein SUMO modification to occur. The SUMO protein family consists of three well accepted members in vertebrates: SUMO1 and the close homologues SUMO2 and SUMO3 with some 50% homology between SUMO1 and SUMO2/3. A fourth family member, SUMO4, has been described though it existence and functional role are still the matter of some debate. Despite their fairly low amino acid sequence identity the SUMO proteins exhibit similar tertiary structures to ubiquitin. Whilst the specific roles of the three SUMO isoforms have not been fully elucidated they have been shown to regulate distinct mitotic processes in mammalian cells. In addition SUMO2 and SUMO3 can form homogenous and mixed chains, with SUMO1, which does not contain the internal SUMO consensus sites (K11) found in SUMO2 and SUMO3, thought to terminate mixed polysumo chains. Protein modification with polysumo chains is involved in processes distinct from those associated with mono-sumoylation. References 1. Kho, C., Lee, A., Jeong, D., Oh, J.G., Chaanine, A.H., Kizana, E, Park, W.J., Hajjar, R.J.SUMO1-dependent modulation of SERCA2a in heart failure. Nature ;477 (7366). PMID: Wilkinson, K. A & Henley, J. M. Mechanisms, regulation and consequences of protein SUMOylation. The Biochemical journal 428, (2010). PMID: Hannoun, Z., Greenhough, S., Jaffray, E., Hay, R. T. & Hay, D. C. Post-translational modification by SUMO. Toxicology 278, (2010). PMID: Gareau, J. R. & Lima, C. D. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nature reviews. Molecular cell biology 11, (2010). PMID: Dou, H., Huang, C., Van Nguyen, T., Lu, L.-S. & Yeh, E. T. H. SUMOylation and de- SUMOylation in response to DNA damage. FEBS letters 585, (2011). PMID: Page 2

3 Assay principle This kit facilitates the fast, effective capture and detection of SUMO modified proteins from biological samples. The kit utilises a high capacity, high specificity SUMO binding matrix together with our easy-to-use purification system for less hands on time and superior performance. Analysis by Western blotting or proteomic methods enables identification and assessment of SUMOylated proteins of interest. Kit contains sufficient SUMO binding matrix to perform 10 assays. Use this kit to: 1. Capture and detect SUMO modified proteins from cell lysates and tissue extracts 2. Demonstrate specific proteins are substrates for SUMOylation in vivo 3. Identify and characterise SUMO modified proteins by proteomic analysis 4. Investigate the role of SUMOylation in specific cellular processes Kit components Reagent Quantity Storage SUMO capture matrix (50% slurry) 400µL 4ºC Spin columns 10 tubes 4ºC Collection tubes 10 tubes 4ºC SUMO1 sheep antibody 10µL -20ºC SUMO2/3 sheep antibody 10µL -20ºC Storage Kit components should be stored at the stated temperatures to ensure stability and activity. SUMO binding matrix should not be refrozen after initial thawing, store at 4ºC. Additional materials required Microfuge tubes (1.5mL) Lysis buffer - 50mM Tris-Cl, ph 7.5, 150mM NaCl, 1% NP-40, 0.5% deoxycholate, 200mM iodoacetamide, 0.1% (v/v) protease inhibitor cocktail III (Roche) Equilibration buffer - 50mM Tris-Cl, ph 7.5, 150mM NaCl, 1% NP-40 Wash buffer - 50mM Tris (ph 7.5), 250mM, NaCl, 1% NP40 Elution buffer (as appropriate) o SDS-PAGE sample loading buffer WB analysis o 0.1% formic acid solution proteomic analysis Target protein specific antibodies WB analysis Reagents for Western blotting Page 3

4 SUMO capture assay Notes: Please read protocol in its entirety before starting. Kit contains sufficient SUMO binding matrix to perform up to 10 assays. Assay preparation Samples Recommend using 200µL lysate at 2mg/mL per assay as a starting point. Adjust lysate concentration with lysis buffer if required. Lysis buffer Assay compatible with a wide range of lysis buffers. Avoid buffer components that cause protein denaturation, especially chaotropes such as urea. Minimise use of reducing agents (e.g. DTT) and detergents where possible. Utilise general cysteine protease inhibitor such as iodoacetamide or N- ethylmaleimide to minimise SUMO deconjugation by SUMO proteases (SENPs) Suggested lysis buffer: 50mM Tris-Cl, ph 7.5, 150mM NaCl, 1% NP-40, 0.5% deoxycholate, 200mM iodoacetamide, 0.1% (v/v) protease inhibitor cocktail III (Roche). Elution buffer Select appropriate elution buffer for intended method of analysis: a) SDS-PAGE sample loading buffer WB analysis b) 0.1% formic acid proteomic analysis Assay optimisation Optimal assay conditions for capture of SUMOylated proteins from specific lysate samples must be determined by the user. Adjustment of the following parameters may facilitate this process: Sample volume, µL Sample concentration, 0.5-2mg/mL SUMO capture matrix volume, 10-50µL settled resin Assay time, 1-4 hours or overnight Page 4

5 Assay protocol 1. Keep reaction components on ice throughout set-up 2. Take Input sample for subsequent analysis 3. Include appropriate controls as required SUMO capture matrix preparation: 4. Resuspend the SUMO capture matrix by gentle inversion of the tube 5. Aliquot 40µL SUMO capture matrix suspension into required number of capped spin columns 6. Add 500µL Equilibration buffer to capped column Mix for 1 minute Remove base cap Centrifuge at low speed ( g, 1 minute) to collect matrix Discard flow through 7. Repeat matrix wash / collection at least twice The SUMO capture assay: 8. Add 200µL lysate to capped SUMO capture matric column and mix by inversion 9. Incubate for 1 hour at 4ºC with rotary mixing 10. Uncap column base and place in a collection tube 11. Centrifuge at low speed ( g, 1 minute) to collect matrix 12. Remove flow through and retain as Unbound Fraction for subsequent analysis if required 13. Replace column in collection tube 14. Wash matrix by adding 500µL Wash buffer to column: Centrifuge at low speed ( g, 1 minute) to collect matrix Repeat three times Elution of captured SUMOylated proteins: 15. For SDS-PAGE / Western blot analysis: Add SDS-PAGE sample loading buffer to capped column and mix by inversion Place column in microfuge tube Heat to 95ºC for 5 minutes Remove base cap Centrifuge at low speed ( g, 1 minute) to collect eluted materials Analyse or store at -20ºC 16. For proteomic analysis: Page 5

6 Add 10 volumes (200µL) 0.1% formic acid to capped column Rotary mix for 5-10 at room temperature Uncap column base and place in microfuge tube Centrifuge at low speed ( g, 1 minute) to collect eluted materials Elution fraction can then be lyophilised and resuspended in trypsin digestion or alternative buffer prior to subsequent processing / analysis, or stored at - 20ºC Western blot analysis This kit includes SUMO1 and SUMO2/3 antibodies for analysis by Western blotting (both sheep polyclonals). Variable SDS-PAGE Samples for analysis SUMO1 antibody SUMO2/3 antibody Secondary antibody Target protein specific antibody (user supplied) Recommendation 10% gel Input Unbound (optional) Elution 1:5000 dilution 1:5000 dilution Donkey anti-sheep (HRP-conjugate) Western blotting conditions must be determined by the user and the antibody applied in conjunction with an appropriate secondary antibody Example Western blotting data Image: Western blot analysis of SUMO capture of SUMO2/3 modified proteins from HeLa nuclear extract. SUMO2/3 conjugates present in 'Input' (1) and Elution (2) samples were detected by SUMO2/3 sheep antibody at 1:1000 dilution Page 6

7 NOTES Page 7

8 Use of product Unless otherwise stated in our catalogue or other company documentation accompanying the product(s), our products are intended for Research Use Only (RUO) and are not to be used for any other purpose, which includes, but is not limited to, unauthorised commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. Warranty Badrilla Ltd makes no warranty of any kind, expressed or implied, which extends beyond the description of the product in this brochure, except that the material will meet our specifications at the time of delivery. Badrilla Ltd makes no guarantee of results and assumes no liability for injuries, damages or penalties resulting from product use, since the conditions of handling and use are beyond our control. Buyer s exclusive remedy for nonconforming products is limited to replacement of or refund for the non-conforming product(s). Badrilla Ltd Leeds Innovation Centre 103 Clarendon Road Leeds LS2 9DF, UK Web: info@badrilla.com Tel: +44 (0) Fax: +44 (0) Version 4 Page 8