Saponin Lysis of RBCs-PDF

Abstract

Lyse red blood cells while leaving the Plasmodium falciparum parasite intact with its parasite membrane and parasitophorous vacuole membrane. Typically used right before freezing down parasites for genomic DNA extraction, or for getting rid of hemoglobin right before running a Western Blot on parasite extracts.

Reagents

• 0.15% Saponin in PBS
• 1X PBS
• 12 mL of Plasmodium falciparum blood culture at 4% hematocrit

Equipment

• Refrigerated centrifuge capable of holding 15 mL conical tubes
• Microcentrifuge

Procedure

1. Centrifuge parasite culture at 1400 rpm (~484xg) for 3 minutes.
2. Aspirate media.
3. Resuspend pellet in 1 ml of 0.15% Saponin (in aliquots in freezer) and transfer to a 1.5 ml eppendorf tube.
4. Incubate for 5 minutes on ice and vortex each minute.
5. Spin at 6000 rpm for 3 minutes at 4 degrees C.
6. Wash with 1 ml 1X PBS (spin at 6000 rpm for 3 minutes) 3 to 4 times.
   1. Each wash step consists of aspirating the supernatant and resuspending the pellet with new wash buffer, before centrifuging.
7. Aspirate off last wash.
8. Store at -20 degrees C until ready to use.