Hi everyone,

I’m working on the removal of lipids co-purified with a soluble bacterial protein complex (E. coli) to prepare it for cryo-EM.

🔬 Workflow and issue:

• The complex is purified using a two-step affinity chromatography protocol: first Ni-NTA, then Strep-Tactin.
• Despite this, the eluate still contains vesicle-like lipidic material, visible in negative staining EM.
• I tried solvent-free delipidation using activated silica gel (ASG) as described in Dolui & Vijayaraj (3 Biotech, 2020): Ratio 1:2 (w/v), i.e. 3.25 g of activated silica for 6.5 mL of sample Incubated at 4 °C for 30 minutes, gentle agitation Protein ~1 mg/mL, in buffer with MOPS, NaCl, and biotin.
• ➡️ Massive loss of the complex (>95%) — I recovered almost nothing, likely due to non-specific binding to the silica

❓ I’d really appreciate insights on:

Thanks in advance for any feedback, shared experiences, or even negative results — everything is helpful 🙏 Happy to give more details if needed!