DIBMA Protocol

Materials:

• Sokalan CP9 (DIBMA) in alkaline solution
• 2 L target buffer (e.g., 50 mM Tris, 200 mM NaCl, pH 7.4)
• 5-mL microdialysis capsule (e.g., QuixSep from Carl Roth)
• 3.5-MWCO dialysis membrane
• 0.22-μm syringe filter
• refractometer
• cell lysate or membrane fraction

Procedures:

1. Dialyze 3 mL DIBMA in a 5-mL microdialysis capsule using a 3.5-MWCO dialysis membrane for 16 h against 1 L target buffer.
2. Exchange buffer and dialyze again for 8 h. The volume will increase by about threefold. Check pH.
3. Filter dialyzed DIBMA stock through a 0.22-μm syringe filter.
4. Determine DIBMA concentration by refractometry using dn/dc = 1.346 M-1 , where c is DIBMA concentration based on Mn.
5. Add DIBMA solution to cell lysate or membrane fraction and incubate at room temperature under gentle agitation for 16 h. Typical conditions are 2.5% (w/v) DIBMA for 42.5 mg mL-1 (wet mass/volume) E. coli membrane fraction.
6. Remove unsolubilized membrane fragments by centrifugation at 100,000 g for 60 min (optional).
7. DIBMALPs containing tagged protein can be subjected to further downstream purification.
8. For SDS-PAGE, first extract polymer by CH3OH/CHCl3/H2O to avoid excessive smearing.
9. For detailed instructions, see Oluwole et al., Angew. Chem. Int. Ed. 2017, 56, 1919–1924 (in particular, Supporting Information).