Materials and Reagents

1. Cellulose nitrate 0.45 µm filter papers (Sartorius, catalog number: 11306-47-N )
   
2. 1.2 µm glass microfiber filters (GE Healthcare, catalog number: 1822-070 )
   
3. 10 ml plastic syringes, Luer slip BD Plastipak (BD, catalog number: 302188 )
   
4. 25 G needle (BD, catalog number: 300600 )
   
5. 1 ml plastic syringes, Luer slip BD Plastipak (BD, catalog number: 303172 )
   
6. 1.5 ml microcentrifuge tubes
   
7. Amicon centrifugal concentrating tubes 4 ml MWCO 10 kDa (Merck, catalog number: UFC801096 )
   
8. Phosphate buffered saline (PBS) made up from Dulbecco A tablets (137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.3) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: BR0014G )
   
9. Ultrapure water (> 18 MΩ cm) for buffer preparations
   
10. Bolt^® Bis-Tris 4-12% precast gels (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NW04120BOX )
    
11. BlUeye prestained protein markers (Geneflow, catalog number: S6-0024 )
    
12. Quick Coomassie Stain (Generon, catalog number: GEN-QC-STAIN-3L )
    
13. Ethanol absolute (200 Proof)
    
14. Nitric acid (HNO₃), 70%
    

Equipment

1. Reusable bottle top filtration device (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: DS0320-5045 )
   
2. Vacuum pump such as KNF Neuberger Vaccum Pump (KNF Neuberger, catalog number: 049268/018121 )
   
3. 500 ml clear Duran bottles (Duran, catalog number: GL 45 )
   
4. Liquid Chromatography system, with a 2 ml injection loop, and a fraction collector; we use the ÄKTA pure 25 L with an F9-R fraction collector (GE Healthcare, model: ÄKTA pure 25 L )
   
5. Size exclusion chromatography column; we use a Superdex 200 Increase 10/300 GL column, bed volume 24 ml (GE Healthcare, catalog number: 28990944 )
   
6. Benchtop refrigerated micro-centrifuge capable of 14,000 x g (e.g., Eppendorf, model: 5418 R )
   
7. Far-UV spectrophotometer with a bandwidth < 1.8 nm and quartz cuvettes. We use a single-beam Beckman DU 800 instrument with microcells that allow measurements with volumes of 50 to 100 μl (Beckman Coulter, model: DU^® 800 )
   
8. A far-UV circular dichroism (CD) spectrometer with a temperature controlled cell holder. We use an AVIV Model 215 instrument (Aviv Biomedical, model: Aviv Model 215 ) with a single cell Peltier controlled cell holder, or make use of the Module B end-station spectrophotometer at the B23 Synchrotron Radiation CD (Diamond Light Source, model: B23 ) Beamline at the Diamond Light Source (Jávorfi et al., 2010; Cole et al., 2016). Alternative instruments are available from Applied Photophysics Ltd (Leatherhead, U.K.), JASCO Inc. (Easton, MD), and Olis Inc. (Bogart, GA)
   
9. Strain free sealable quartz cuvettes of appropriate path length fitting the CD instrument’s cell holder. We use Teflon stoppered Hellma Suprasil cells of various thickness, mostly 0.1-cm
   
10. A well-ventilated chemical fume hood should be accessible for cleaning cuvettes with HNO₃
    

Software

1. An analysis software that can import the CD data files and allows curve fitting to a user defined set of equations is required. We use Origin version 7.5 and later (OriginLab Corp., Northampton, MA), but many other programs. e.g., Igor Pro, MATLAB, Micromath Scientist, SigmaPlot, etc. will work

Procedure