Methodology for the Extraction of Plasmodium Protein. Learning Objectives:

Transcription

1 Proteomics Extraction of Plasmodium Protein Methodology for the Extraction of Plasmodium Protein Extraction of entire protein from the sample requires an optimized protocol and many protocols have been developed to increase the amount of protein in the extract. Learning Objectives: After interacting with this learning object, the learner will be able to: Understand the steps involved in production and maintenance of parasite culture. Summarize protein extraction from protein culture. Interpret the results of the experiment. Assess the troubleshooting steps at various stages of the experiments. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.

2 Blood Processing Wet the cotton with spirit and with it clean the area of the hand where the blood is to be drawn. Using a sterilized needle and syringe, take out blood from the B blood group Rh +ve individual carefully under proper supervision. Discard the tip into a discard bin. Now transfer the collected blood into a 10% CPDA bag containing Citrate, Phosphate, Dextrose and Adenine anticoagulant. The sample can now be stored for about 6months at -80 C.

3 Blood Processing Shake the CPDA bag containing blood to mix the components. Using a syringe, take out the required amount of blood and transfer it to an autoclaved appendorf. Centrifuge the tube at 2400 rpm, 4 C for 5minutes to separate the RBCs. Discard the yellow colored supernatant along with a little bit of blood to remove WBCs. Use the pellet containing RBCs for the process ahead.

4 Parasite Processing Take out the vial containing the frozen parasite culture and gradually thaw it at 37 C in a incubator.

5 Parasite Processing Clean the surface of the balance with a tissue paper. Place a butter paper and tare the weight of the paper.

6 Parasite Processing Weigh 1.2 g of NaCl and dissolve in 10ml of water to make 12% NaCl. Similarly, weigh 0.16 g of NaCl and dissolve in 10ml of water to make 1.6% NaCl.

7 Parasite Processing Add 200 μl of 12% NaCl drop wise to the tube containing the parasite and incubate at room temperature for 2 minutes. Following incubation, transfer the contents of the tube to a falcon tube. Now add 8ml of 1.6% NaCl to the tube. Centrifuge the contents at 2600 rpm, room temperature for 5minutes.

8 Parasite Processing The supernatant is observed in red color owing to the lysis of the RBC and release of heamoglobin.

9 Parasite Processing Clean the surface of the balance with a tissue paper. Place a butter paper and tare the weight of the paper.

10 Parasite Processing Weigh 0.45g of NaCl and dissolve it in 50 ml of distilled water. Mix the contents thoroughly.

11 Parasite Culturing Add 8ml of 0.9% NaCl solution and centrifuge the contents for 5minutes at room temperature and 2400 rpm. Discard the supernatant obtained. To the pellet add 5ml of supplemented media. This is done to wash the pellet with media. Vortex the tube and centrifuge for 5minutes at 2400 rpm and room temperature.

12 Parasite Culturing Add 0.25ml of RBC and 4.75ml of supplemented media and transfer it to a 5ml culture flask. Incubate the flask at 37 C for 4hours.

13 Parasite Culturing Without disturbing the media, take out about 1ml of the culture media and centrifuge the contents at 2400 rpm for 5minutes and room temperature.

14 Parasite Culturing Discard the supernatant. To the pellet, add 1 ml of sorbitol and incubate for 5minutes. This step is necessary to synchronize the cells at the same stage.

15 Parasite Culturing Reconstitute the pellet in media containing 3% hematocrit. Transfer the contents to a culture flask and place it in a candle jar. Incubate at 37 C with the candle lit.

16 Parasite Culturing Take a smear of the culture and stain it using field s stain to identify the ring stage of the parasite. If the ring stage is clearly observable then the user can proceed to the next stage, else the synchronization step is to be performed again.

17 Parasite Culturing Transfer the synchronous culture to a 30 ml flask containing RPMI 1640 media. The culture is allowed to grow till 9% parasitemia is reached.

18 Parasite Culturing After 9% parasitemia is achieved, pipette out the media into a falcon tube. Centrifuge the contents for 5minutes at 2600 rpm. Discard the supernatant. To the pellet add PBS and saponin and keep the tube on ice for 5minutes.

19 Parasite Culturing The cells are now lysed and the solution becomes colorless. The parasites as a result of this are released.

20 Parasite Culturing Vortex the tube and then centrifuge the contents for 5minutes. This leads to separation of the contents into two phases.

21 Parasite Culturing Discard the supernatant. Add 1ml of PBS to the pellet and vortex it thoroughly. Centrifuge the contents at 16000g, 4 C for 5minutes. Repeat the step till a white pellet is observed.

22 Parasite Culturing Store the pellet at -80 C until protein extraction is done.

23 Reagents Preparation Clean the surface of the balance. Place a butter paper and tare the weight of the paper.

24 Reagents Preparation Weigh the required amount of Urea and dissolve in water using a magnetic stirrer. Take urea solution into an appendorf tube using a pipette. Add protease inhibitor to the urea solution and mix thoroughly by vortexing the tube.

25 Protein Extraction Sonicate the sample by providing 6 cycles for 5 seconds, 20% amplitude and 59 seconds gap. The process is to be done entirely on ice. Sonication helps in cell lysis by the use of ultrasound waves.

26 Protein Extraction Vortex the sonicated protein sample thoroughly for proper mixing.

27 Protein Extraction Add CHAPS to the sonicated sample. Shake the sample intermittently for 20 minutes. Now centrifuge the contents at 1600g, 4 C for 30 minutes. Separate the supernatant and pellet into two tubes and store both the tubes at -80 C. The sample is now ready for further use.

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