Purification and TEM of Afp and Its Variants

Materials and Reagents

Note: All solutions, unless otherwise specified, were prepared with distilled deionised Millipore-microfiltered (MilliQ) water, autoclaved for 15 min at 1.1 kg/cm² at 121 °C and stored at room temperature. If antibiotics or supplements were to be added, the medium was cooled to a temperature of 50-60 °C before addition of antibiotic or supplement solution. The following antibiotics were used at stated concentrations (μg/ml): ampicillin, 100; chloramphenicol, 30; and spectinomycin, 100.

1. E. coli strains containing afp genes under arabinose promoter
   Note: Please refer to the original manuscript (Rybakova et al., 2013) for strains and antibiotic resistance description.
   
2. LB-agar (Merck KGaA, catalog number: VM459383236 )
   
3. DNase (Roche Diagnostics, catalog number: 04716728001 )
   
4. 0.7 % filtered (pore size 0.2 um) uranyl acetate diluted in sterile water (pH 5.0) (Electron Microscopy Sciences, catalog number: 22400 )
   
5. Sephacryl S-400 HR resin (GE, http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17395/17060901)
   
6. 20% L-Arabinose (Acros organics) (Thermo Fisher Scientific, catalog number: AC104985000 ) (see Recipes)
   
7. 0.4x Luria-Bertani broth (LB) (Life Technologies, catalog number: 12795-027 ) (see Recipes)
   
8. 0. 2% sodium azide (Sigma-Aldrich, catalog number: S8032 ) (see Recipes)
   
9. 250 mM TBS buffer (see Recipes)
   
10. TM buffer (see Recipes)
    

Equipment

1. Spectrophotometer
   
2. Orbital mixer incubator (Raytek Corporation)
   
3. 1.7 ml microcentrifuge tubes
   
4. Eppendorf centrifuges
   
5. Sanyo soniprep 150 sonicator (18 Ω) (Panasonic Corporation)
   
6. 0.45-µm-pore-size cellulose acetate filters (Advantec MFS, catalog number: 03CP045AN )
   
7. 50 ml BD Falcon Tubes
   
8. Sephacryl S-400 HR beads (GE Healthcare, catalog number: 17-0609-10 ) for size exclusion chromatography (SEC)
   
9. The SEC column with a bed volume of 1.5 by 46 cm (Bio-Rad Laboratories, model: 737-4155 )
   
10. 1.5 x 50 cm (D x L) column (Farmacia)
    
11. BioLogic LP system (Bio-Rad Laboratories, model: 731-8350 )
    
12. Beckman coulter Optima TM L-100K ultracentrifuge (Beckman Coulter, model: 392050 )
    
13. Polaron E5100 SEM coating unit
    Note: This unit is from before 2000 and is no longer available, however a similar unit that is equivalent can be found at http://www.quorumtech.com/products/glow-discharge-system-free-standing.html.
    
14. Whatman #1 filter paper (Whatman)
    
15. Plastic-coated 200-mesh grid (ProSciTech, catalog number: GCU200 )
    
16. Morgagni 268D TEM (FEI)
    
17. Megapixel III digital camera
    Note: The catalog numbers and model numbers are not important for reproducibility.
    
18. Polypropylen tubes used for the size exclusion chromotography (polypropylene 12 x 75 mm, 5 ml) (Labserv, catalog number: LBS504N )
    
19. Polypropylen tubes for ultracentrifugation (polycarbonate centrifuge bottles with cap assembly 16 x 76 mm, capacity 10.4 ml) (Beckman Coulter, catalog number: 355603 )
    
20. Falcon tubes (Thermo Fisher Scientific, catalog number: 14-432-22 )
    
21. Protect microorganism preservation system (Technical Service Consultants) for preservation of E. coli cells at -80 °C
    Note: When required, a single inoculated bead was removed from the vial and dropped into a LB broth containing the appropriate antibiotic.
    

Procedure