Saponin Lysis of RBCs

Curators

Jingyang Chen, Seattle Biomedical Research Institute, 307 Westlake Ave N, Suite 500, Seattle, WA, 98109, USA. jingyang.chen@sbri.org

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Abstract

Lyse red blood cells while leaving the Plasmodium falciparum parasite intact with it's parasite membrane and parasitophorous vacoule 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 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.

Critical steps

Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.

Troubleshooting

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Notes

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References

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