Materials and Reagents

1. Bacteria: Gram negative Escherichia coli (E. coli) or yeast Saccharomyces cerevisiae (S. cerevisiae)
   Notes:
   Amyloids exist in E. coli and S. cerevisiae. In both organism amyloids can be functional. In E. coli the most well know example is the curli protein which is assembled in the extracellular medium and allow the attechemnet of the bacteria to the surface and sustain the formation of biofilms (Chiti and Dobson, 2006; Chapman et al., 2002).
   In S. cerevisiae, amyloids confer to cells selctive advantage under certain physiological conditions and they aren’t harmful (Chien et al., 2004; Shorter and Lindquist, 2005; Wickner et al., 2007), a few examples are Sup35p y Ure2p, Rnq1p. The conversion of Sup35 from soluble to the amyloid state drives the reduction of translation termination activity (Wickner et al., 1995).
   
2. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 04 693 159 001 )
   
3. Silica beads (1 mm lysing matrix C) (MP Biomedicals, catalog number: 6912-050 )
   
4. Pure agarose D2 (Pronadisa, catalog number: 8034 )
   
5. Detergents: SDS (Bio-Rad Laboratories, catalog number: 161-0301 ) and Sarkosyl 97% N-lauroylsarcosine (Sigma-Aldrich, catalog number: L-9150 )
   
6. PVDF membrane (Bio-Rad Laboratories)
   
7. Antibodies
   Notes:
   Depends on the protein you work, the antibody may be against the protein that you are studying for aggregation. A recommended strategy if the user is going to generate a recombinant protein is to include a tag in the amino or carboxyl terminal of the protein and use a specific antibody against the tag. One example is the Histidine-tag, the corresponding antibody would be a Monoclonal anti-polyhistidine (Sigma-Aldrich, catalog number: H1029 ).
   Antibodies that recognize monomer state may still be able to identify the protein in polymers even is the protein conformation has changed. Usually monomer antibodies have a broad range recognition epitopes. In our hands the antibodies used were:
   For E. coli, house-made polyclonal antibodies against RepA (generated in rabbit immunized with soluble RepA), (Gasset-Rosa et al., 2014); and monoclonal anti poly-histidine against His-RepA (WH1) (Sigma-Aldrich, catalog number: H1029) (Molina-García and Giraldo, 2014). Both of them work.
   For S. cerevisiae, anti-HA antibody (Roche Diagnostics, catalog number: 12CA5 ) against the HA (Human influenza hemagglutinin) tag of Sup35 protein (unpublished).
   
8. Cell culture medium
   
   1. Luria Bertani (LB) medium (see Recipes)
      
   2. Yeast extract peptone dextrose (YPD) (see Recipes)
9. Lysis buffer (see Recipes)
   
10. Running buffer (see Recipes)
    
11. Loading buffer (see Recipes)
    
12. Transference buffer (see Recipes)
    

Equipment

1. Fast Prep-MilliPore/MP FastPrep-24 homogenizer (catalog number: 6004-500 )
   
2. Horizontal agarose electrophoresis system (Electrophoresis power supply EPS601 General Electrics)
   
3. Wet/Tank transfer blotting system (Bio-Rad Laboratories)
   

Procedure