Materials and Reagents

Leishmania donovani (L. donovani) parasites
Glucose (Sigma-Aldrich, catalog number: G8270 )
Hydroxyurea (Sigma-Aldrich, catalog number: H8627 )
RPMI-1640 (Life Technologies, catalog number: 21875-034 )
Fetal Bovine Serum heat inactivated (HIFBS) (Life Technologies, catalog number: 10270-106 )
Penicillin/Streptomycin (Life Technologies, catalog number: 15140-122 )
HEPES buffer (Life Technologies, catalog number: 15630-056 )
Dry ice
Ethanol
E-64 (Sigma-Aldrich, catalog number: E3132 )
CHAPS (Sigma-Aldrich, catalog number: 26680 )
ASB-14 (Sigma-Aldrich, catalog number: A1346 )
DTT (Sigma-Aldrich, catalog number: 43815 )
Bio-Lyte^® 3/10 Ampholyte (Bio-Rad Laboratories, catalog number: 163-2094 )
Urea (Sigma-Aldrich, catalog number: U5378 )
Thiourea (Sigma-Aldrich, catalog number: T7875 )
Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 )
Phosphate Buffered Saline (PBS) (see Recipes)
Lysis Buffer (see Recipes)
Rehydration Buffer (see Recipes)
Fully supplemented medium (see Recipes)

Equipment

Pipette
Centrifuge
Spiramix

Procedure

Leishmania donovani parasites in the logarithmic phase (~5 x 10⁶ parasites/ml) were cultured in fully supplemented culture medium at 25 °C and synchronized with the addition of 2.5 mM hydroxyurea for 12 h.
After this period of time the parasites are gently washed three times with 10 ml of PBS and released in 10 ml of fully supplemented RPMI medium.
5 x 10⁸-10⁹ parasites are harvested by centrifugation (500 x g, 15 min, RT).
The parasitic pellet is washed twice with 10 ml of PBS supplemented with 0.1% (w/v) glucose.
The parasitic pellet is resuspended in 50 μl Lysis buffer using a pipette.
The lysed parasites are subjected to five freeze-thaw cycles in solid CO₂/ethanol.
The sample is centrifuged at 20,000 x g for 30 min at 4 °C.
To the supernatant 400 μl of Rehydration buffer [containing 0.2% (v/v) Bio-Lyte^®] are added.
The sample is incubated for 30 min at RT under gentle rotation.
After the end of the incubation, the sample is centrifuged at 17,000 x g for 40 min at RT.
The supernatant can now be used for proteomic analysis or can be stored at -80 °C for future use.

Notes

Extra caution is required at all steps of the procedure as not the protein sample to be contaminated by environmental proteins (e.g. keratin). Preferably avoid using bare hands during the procedure.
After the addition of Rehydration buffer the sample should not be exposed to temperatures below 10 °C, due to urea-mediated sedimentation.
Bio-Lyte^® should be added just before the use of the Rehydration Buffer.

Recipes

PBS
Dissolve
8 g of NaCl
0.2 g of KCl
1.44 g of Na₂HPO₄
0.24 g of KH₂PO₄
In 800 ml distilled H₂O
Adjust pH to 7.4
Add H₂O to 1 L
Sterilized by autoclaving
Lysis Buffer
400 μl Tris-HCl (pH 9.5)
20 μl 0.5 M EDTA (pH 8.0)
100 μl 1 mM E-64
Add distilled H₂O up to 10 ml
Aliquot and stored at -20 °C
Rehydration Buffer
To a volume of 3 ml distilled H₂O dissolve
4.8 g urea
1.52 g thiourea
0.4 g CHAPS
0.1 g DTT
0.24 g ASB-14
5 μl 0.1% (w/v) bromophenol blue
Add distilled H₂O up to 10 ml
Aliquot and stored at -20 °C
Fully supplemented medium
RPMI-1640
10% (v/v) HIFBS
10 mM HEPES
100 U/ml Penicillin/Streptomycin

Acknowledgments

This protocol was adapted from Clayton (2002) and Smirlis et al. (2006). Partial funding from the General Secretariat of Research and Technology action “Proposals for the development of Research Institutes, Krepis” supported this work.

References

Alexandratos, A., Clos, J., Samiotaki, M., Efstathiou, A., Panayotou, G., Soteriadou, K. and Smirlis, D. (2013). The loss of virulence of histone H1 overexpressing Leishmania donovani parasites is directly associated with a reduction of HSP83 rate of translation. Mol Microbiol 88(5): 1015-1031.
Clayton, C. E. (2002). Life without transcriptional control? From fly to man and back again. EMBO J 21(8): 1881-1888.
Smirlis, D., Bisti, S. N., Xingi, E., Konidou, G., Thiakaki, M. and Soteriadou, K. P. (2006). Leishmania histone H1 overexpression delays parasite cell-cycle progression, parasite differentiation and reduces Leishmania infectivity in vivo. Mol Microbiol 60(6): 1457-1473.