Extraction of Plasmodium Protein - OSCAR Main Pageoscar.iitb.ac.in/onsiteDocumentsDirectory/Extraction of Plasmodium... · • Interpret the results of the experiment. ... Extraction

Proteomics

Extraction of Plasmodium Protein

Methodology for the Extraction of Plasmodium Protein

Extraction of entire protein from the sample requires an optimizedprotocol and many protocols have been developed to increase theamount 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.


Wet the cotton with spirit and with it clean the areaof the hand where the blood is to be drawn.

Using a sterilized needle and syringe, take out bloodfrom the B blood group Rh +ve individual carefullyunder proper supervision.

Discard the tip into a discard bin. Now transfer thecollected blood into a 10% CPDA bag containingCitrate, Phosphate, Dextrose and Adenineanticoagulant. The sample can now be stored forabout 6months at -80°C.

Blood Processing


Shake the CPDA bag containing blood to mix thecomponents. Using a syringe, take out the requiredamount of blood and transfer it to an autoclavedappendorf.

Centrifuge the tube at 2400 rpm, 4°C for 5minutes toseparate the RBCs.

Discard the yellow colored supernatant along with alittle bit of blood to remove WBCs. Use the pelletcontaining RBCs for the process ahead.

Blood Processing


Take out the vial containing the frozen parasiteculture and gradually thaw it at 37°C in a incubator.

Parasite Processing


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

Parasite Processing


Weigh 1.2 g of NaCl and dissolve in 10ml of water tomake 12% NaCl.

Similarly, weigh 0.16 g of NaCl and dissolve in 10ml ofwater to make 1.6% NaCl.

Parasite Processing


Add 200 μL of 12% NaCl drop wise to the tubecontaining the parasite and incubate at roomtemperature 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, roomtemperature for 5minutes.

Parasite Processing


The supernatant is observed in red color owing to thelysis of the RBC and release of heamoglobin.

Parasite Processing


Clean the surface of the balance with a tissue paper.

Place a butter paper and tare the weight of the paper.

Parasite Processing


Weigh 0.45g of NaCl and dissolve it in 50 ml ofdistilled water.

Mix the contents thoroughly.

Parasite Processing


Add 8ml of 0.9% NaCl solution and centrifuge thecontents for 5minutes at room temperature and 2400rpm.

Discard the supernatant obtained.

To the pellet add 5ml of supplemented media. This isdone to wash the pellet with media. Vortex the tubeand centrifuge for 5minutes at 2400 rpm and roomtemperature.

Parasite Culturing


Add 0.25ml of RBC and 4.75ml of supplemented mediaand transfer it to a 5ml culture flask. Incubate theflask at 37°C for 4hours.

Parasite Culturing


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

Parasite Culturing


Discard the supernatant. To the pellet, add 1 ml ofsorbitol and incubate for 5minutes. This step isnecessary to synchronize the cells at the same stage.

Parasite Culturing


Reconstitute the pellet in media containing 3%hematocrit.

Transfer the contents to a culture flask and place it ina candle jar.

Incubate at 37°C with the candle lit.

Parasite Culturing


Take a smear of the culture and stain it using field’sstain to identify the ring stage of the parasite. If thering stage is clearly observable then the user canproceed to the next stage, else the synchronizationstep is to be performed again.

Parasite Culturing


Transfer the synchronous culture to a 30 ml flaskcontaining RPMI 1640 media. The culture is allowed togrow till 9% parasitemia is reached.

Parasite Culturing


After 9% parasitemia is achieved, pipette out themedia into a falcon tube.

Centrifuge the contents for 5minutes at 2600 rpm.Discard the supernatant. To the pellet add PBS andsaponin and keep the tube on ice for 5minutes.

Parasite Culturing


The cells are now lysed and the solution becomescolorless. The parasites as a result of this arereleased.

Parasite Culturing


Vortex the tube and then centrifuge the contents for5minutes. This leads to separation of the contents intotwo phases.

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.

Parasite Culturing


Store the pellet at -80°C until protein extraction isdone.

Parasite Culturing


Clean the surface of the balance.

Place a butter paper and tare the weight of the paper.

Reagents Preparation


Weigh the required amount of Urea and dissolve inwater using a magnetic stirrer.

Take urea solution into an appendorf tube using apipette.

Add protease inhibitor to the urea solution and mixthoroughly by vortexing the tube.

Reagents Preparation


Sonicate the sample by providing 6 cycles for 5seconds, 20% amplitude and 59 seconds gap. Theprocess is to be done entirely on ice. Sonication helpsin cell lysis by the use of ultrasound waves.

Protein Extraction


Vortex the sonicated protein sample thoroughly forproper mixing.

Protein Extraction


Add CHAPS to the sonicated sample.

Shake the sample intermittently for 20 minutes.

Now centrifuge the contents at 1600g, 4°C for 30minutes.

Separate the supernatant and pellet into two tubesand store both the tubes at -80°C. The sample is nowready for further use.

Protein Extraction