Materials and Reagents

Note: All chemicals are stored in dedicated rooms and safety cabinets. They are manipulated in accordance with product safety data sheets (SDS) and manufacturer instructions were followed for shelf-life (see below for storage temperatures–RT = Room Temperature).

Consumables:

1. 1 ml syringes (Terumo, catalog number: SS+01T1 )
2. 26 G ½” needles (Terumo, catalog number: AN-2613R1 )
3. Transfer pipettes (rod volume 3 ml) (Biosigma, catalog number: 390514 )
4. Microcentrifuge tubes (1.5 ml SafeLock) (Eppendorf, catalog number: 7953691744 )
5. Polypropylene 15 ml centrifugation tubes (Sarstedt, catalog number: 62.554.502 )
   
   

1. Parasites and Mice
   1. Plasmodium berghei ANKA parasites (kind gift from Dr. O. Silvie, CIMI, Paris, stored in liquid nitrogen, diluted in room temperature RPMI post-thawing prior to being i.p.-injected to mice)
   2. CD-1 mice (25-30 g male CD-1^® IGS, Charles River)
      Mice are i.p. infected with 10⁶ infected red blood cells and parasitemia is monitored using thin blood smears. For the procedure detailed below, parasitemia of infected mice should be between 5% and 10%. This is usually obtained between day 5 and 10 post infection.
   3. Sodic Heparin (25,000 UI/5 ml, Sanofi, stored at RT)
      
      
      
2. iRBC (infected Red Blood Cell) sequential lysis
   1. Tris Base (Euromedex, catalog number: 200923-A , stored at RT)
   2. KCl (Sigma-Aldrich, catalog number: 60128 , stored at RT)
   3. CaNa₂·EDTA (Sigma-Aldrich, catalog number: ED2SC , stored at RT)
   4. NaCl (Euromedex, catalog number: 1112 , stored at RT)
   5. MgSO₄ (Sigma-Aldrich, catalog number: M7506 , stored at RT)
   6. Triton X-100 (Euromedex, catalog number: EU2000-B , stored at RT)
   7. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 , stored at RT)
   8. Nycodenz^® powder (Eurobio Ingen, catalog number: 1002424 , stored at RT)
   9. 10x PBS (Euromedex, catalog number: ET330-A , stored at RT)
   10. cOmplete^TM EDTA-free anti-protease cocktail (Roche, catalog number: 5056489001 , stored at 4 °C)
   11. Percoll solution (GE Healthcare, catalog number: 17-0891-01 , stored at 4 °C)
   12. RPMI medium 1640 w/HEPES (Thermo Fisher Scientific, catalog number: 12017599 , stored at 4 °C)
   13. Equinatoxin II enzyme (kind gift from Prof.Gregor Anderluh, National Institute of Chemistry, Ljubljana, Slovenia, aliquots are stored in PBS at -80 °C)
   14. Saponin lysis solution (Sigma Aldrich, catalog number: S7900 , powder stored at RT)
   15. 60% Nycodenz solution (see Recipes for storage conditions)
       Nycodenz buffered medium
       Nycodenz stock solution
       60% Nycodenz solution (freshly prepared)
   16. Equinatoxin II (EQT) lysis buffer (see Recipes)
   17. Saponin lysis solution (see Recipes)
       Saponin 10x stock solution
       1x Saponin lysis solution (freshly prepared)
   18. Triton lysis buffer (see Recipes)
   19. Percoll gradient layers (see Recipes)
       90% Percoll stock solution
       80% Percoll layer
       70% Percoll layer
       60% Percoll layer
       40% Percoll layer
       
       
       
3. SDS-Page and Western blot analysis
   1. Tris Base (Euromedex, catalog number: 200923-A , stored at RT)
   2. SDS 20% solution (Sigma-Aldrich, catalog number: 0 5030 , stored at RT)
   3. Glycine (Euromedex, catalog number: 26-128-6405 , stored at RT)
   4. Glycerol (Euromedex, catalog number: EU3550 , stored at RT)
   5. Tween-20 (Sigma-Aldrich, catalog number: 8221840500 , stored at RT)
   6. Polyacrylamide gels (Bio-Rad Mini-PROTEAN^® TGX^TM Precast Gels, catalog number: 4561043 , stored at 4 °C
   7. PageRuler Prestained Protein Ladder (Thermo Scientific, catalog number: 26619 , stored at -20 °C)
   8. Low fat milk powder (Régilait, skimmed milk 0.8% fat, stored at RT)
   9. Western blot detection kit (SuperSignal^TM West Dura Extended Duration Substrate, Thermo Fisher Scientific, catalog number: 34075 , stored at 4 °C)
   10. Antibodies (primary and secondary antibodies are listed in Table 1)
   11. 3x Sample buffer (see Recipes)
   12. Running buffer (see Recipes)
   13. Transfer buffer (see Recipes)
   14. Blocking solution (see Recipes)
   15. Western blot washing buffer (see Recipes)
       
       Table 1. List of primary and secondary antibodies used in this study
       
       ^aThe Plasmodium antigens are from P. yoelii (EXP2), or P. falciparum (Aldolase, HSP70). However, the antiserum raised against these antigens cross react with P. berghei EXP2, Aldolase and HSP70 respectively. The antibody targeting human PP1c also recognizes mouse PP1c.
       

Equipment

1. Laboratory rotating wheel (Stuart, model: SB3 )
2. 50 ml Nalgene Oak Ridge High-Speed Centrifuge Tubes with sealing cap, PSF (Thermo Fisher Scientific, catalog number: 3137-0050 )
3. Refrigerated centrifuge for microtubes (Eppendorf, model: 5427R )
4. Centrifuge for 15 ml tubes (Eppendorf, model: 5810R )
5. Superspeed centrifuge (Sorvall Lynx 4000 centrifuge with fixed angle rotor F20-12x50 LEX, ThermoScientific)
6. Sonication device (Bioruptor plus, Diagenode, catalog number: B01020001 )
7. Block heater (Stuart, model: SBH200D )
8. Gel electrophoresis and transfer chamber (Bio-Rad Miniprotean Tetra Cell System)
9. Filter paper for Western blot transfer (Whatman 3MM CHR, GE Healthcare, catalog number 3030-917 )
10. Nitrocellulose membrane (Amersham protran Premium 0.45 Nitrocellulose, Thermo Fisher Scientific, catalog number: 15269804 )
11. Vacuum sealer (KitchenGynti)
12. Rocker platform (Labnet reciprocal, 3D or rocker shaker)
13. Molecular Imager (Bio-Rad ChemiDoc XRS+)
    

Software

1. Image Lab 5.0 (Bio-Rad)

Procedure