Isolation of Inner Membrane Vesicles from Escherichia coli by Using an Affinity Tag

Materials and Reagents

1. Isopropyl β-D-1-thiogalactopyranoside (IPTG)
   
2. Medium copy plasmid pLP8 for recombinant protein expression (Plac lacIq KanR, Li and Young, 2012)
   
3. Bacto Tryptone (BD Biosciences, catalog number: 211705 )
   
4. Halt protease inhibitor (Thermo Fisher Scientific, catalog number: 78425 )
   
5. RNase A (Sigma-Aldrich, catalog number: R5503 )
   
6. DNase I (Thermo Fisher Scientific, catalog number: NC9709009 )
   
7. anti-FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220 )
   
8. Poly-Prep chromatography column (Bio-Rad Laboratories, catalog number: 731-1550 )
   
9. Micro BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23235 )
   
10. Triton X-100
    
11. Tween 20
    
12. Tryptone medium
    
13. Phosphate buffered saline (PBS) (see Recipes)
    
14. Phosphate buffered saline with Tween 20 (PBST) (see Recipes)
    

Equipment

1. Centrifuges for 1.5 ml, 10 ml and 100 ml volumes
   
2. High shear fluid processor (Microfluidics, model: LV1 ) or French press
   

Procedure