Related LOs: Culture handling, Blood properties > Prior Viewing – IDD:6 Serum extraction, IDD:1 Bacterial extraction > Future Viewing –IDD: 11 Protein

Related LOs: Culture handling, Blood properties > Prior Viewing – IDD:6 Serum extraction, IDD:1 Bacterial extraction > Future Viewing –IDD: 11 Protein quantification, IDD: 15 Isoelectric focusing, IDD: 18 Second dimension separation, IDD: 20 Staining

Course Name: Plasmodium Protein Extraction Level(UG/PG): UG Author(s): Dinesh Raghu, Vinayak Pachapur Mentor: Dr. Sanjeeva Srivastava

*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license

Methodology for the extraction of Plasmodium protein

Extraction of the entire protein from the sample requires optimized protocol and many protocols have been

developed to increase the protein amount in the extract

Learning objectives

After interacting with this learning object, the learner will be able to:

1. Define steps involved in production and maintenance of parasite culture

2. Identify the steps involved for maintaining parasite culture

3. Summarize protein extraction from the plasmodium culture

4. Interpret the results of the experiment.

5. Assess the troubleshooting steps involved in the experiments.

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Master Layout

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1Blood processing (Slide:5-7)

Reagent preparation (Slide:30-32)

Protein extraction (Slide:33-35)

Parasite processing (Slide:15-29)

Display a image from each of these steps, with user click.

Parasite processing (Slide:8-14)

Definitions and Keywords

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11. Protein : are the biomolecules, composing of amino acids, forming the building block of the

system and performs most of the biological functions in the system

2. Protein extraction: The process by which the proteins from the cell are recovered for analysis

purpose is called protein extraction. The chemicals involved in the extraction are

3. Parasetima: Number of infected RBCs per 100 RBCs

4. Supplemented media: RPMI 1640 medium with 25mM HEPES (pH 7.5), 25mM NaHCO3

, 50mg/liter hypoxanthine, 2g/L glucose (Sigma G7021), 10% human B Rh+ve blood plasma and

40μg/ml gentamicin sulfate

5. CHAPS: 3-{Dimethyl[3-(4-{5,9,16-trihydroxy-2,15-dimethyltetracyclo[8.7.0.02,7.011,15]

heptadecan-14-yl}pentanamido)propyl]azaniumyl}propane-1-sulfonate (“CHAPS”) is a constituent

of rehydration buffer is a zwitterionic detergent that is used to solubilize the proteins including

membrane proteins.

Step 1:

Audio Narration Description of the action

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3Animator should show a needle syringe, piercing the hand to draw blood out. Collect 10ml of blood from the individual. The user should click on the piston of syringe to collect the blood.Animate, user transferring the collected blood into CPDA bag as shown in figure and the blood should be filled in the bag.

Collect the blood from the B-blood group (Rh +ve) individual in the 10% CPDA bag containing Citrate, phosphate, Dextrose and adenine anticoagulants to preserve whole blood for 35days.

T1: Blood processing

CPDA Bag

Step 2: T1: Blood processing

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Centrifugerotor

Video File: Centrifuge

Step 2:


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3Animate user shaking the cdpa bag and take out the collected blood from the bag into centrifuge tubes. The animator should draw a centrifuge as shown in previous slide. Instruct user to open the lid of centrifuge and rotor. Zoom in the rotor, place the tube, balance equal number of tubes inside the rotor. Close the lid of rotor and of centrifuge with hand action. Instruct user to set the rpm, temperature and time parameters, along with display. User can increase and decrease the values of set parameters (set as in right hand side). Animate the clock for 5min. Once 5 minutes are over user should open the lid by click in “open” button and remove the tube out.

Please include the buttons like enter, set, start, open on the centrifuge display.

Shake the blood containing in buffer bag well and centrifuge at 2400 rpm for 5min for the RBC to settle down. Preserve the RBC's for later stages.

T1: Blood processing

RBC

Step 3:


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3 Animate like the user taking a tube labeled as “parasite” from -80’C fridge by opening the freezer and taking out the tube and keep it in the 37’C incubator by opening it.

Draw -80’C fridge and 37’C incubator an small tube labeled as”parasite” as shown in fig

Show a clock running 15 minutes

Place the frozen parasite culture at 37’C and allow it to thaw, to be used later in the experiemnt.

T2: Parasite processing

-80’C 37’C

Step 4:

Audio Narration (if any)

Description of the action

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Show a measuring balance. A hand action when clicked by user should ON the Instrument, pick paper from rack, place it on the balance so that balance reads 0.03g and the user should press ”0” on the balance to make the reading to “0.00”. This action need to be performed for each reagent weighing.

Clean the surface of the balance, Tare the weight of the paper before weighing.

Video File: balancing

Step 5 :


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3Show the weigh balance, spatula, NaCl bottle, water bottle, measuring cylinder. click on the NaCl bottle and spatula to measure 12g of NaCl and 1.6g of NaCl separately in two bottles.Animate like the user pouring water to the cylinder (zoom and show the measurement) and the volume should increase till 100 ml and then click on the cylinder to pour the water to the weighed 12g NaCl and follow the same for the 1.6g NaCl. Animate user, shaking the bottles for mixing.

Prepare 12% and 1.6% NaCl for RBC lysis.

NaCl


Step 6:


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3Animator should draw 2 bottles labeled as 12% NaCl and 1.6% NaCl the user should click on it.

Draw a ink filler and the user should press on it to take the 12% NaCl.

Animate like when ever user clicks on the filler a drop of solution should fall in to the tube labeled as parasite (taken out from the 37’C incubator)Show like keeping the sample in room temperature and clock running for 2 minutes followed by addition of 1.6% NaCl like earlier steps.Perform the centrifugation as discussed earlier (slide 7)

Add 12 % NaCl drop wise and incubate for 2 mins at room temperature and add 1.6% NaCl and centrifuge at 2400 rpm for 5min


Step 7:


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3 Animator should show the content in the tube slowly changing from colorless solution to red color

Animate like the colorless solution turning to red color, with slow increase in color density with time.

RBC are lysed and the parasites are released. RBC lyses results in the red color of the solution.


Step 8:



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Show a measuring balance. A hand action when clicked by user should ON the Instrument, pick paper from rack, place it on the balance so that balance reads 0.03g and the user should press ”0” on the balance to make the reading to “0.00”. This action need to be performed for each reagent weighing.


Step 8 :


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3Let user takes out weigh balance, spatula, NaCl bottle, water bottle, measuring cylinder from the rack. click on the bottles and spatula to measure 0.9g of NaClAnimate like the user pouring water to the cylinder (zoom and show the measurement) the volume should increase till 100 ml and then click on the cylinder to pour the water to the weighed 0.9g NaCl. Animate user, shaking the tube for proper mixing, to dissolve the reagent into the solution.

Prepare 0.9% NaCl reagent for the parasite processing.

NaCl


Step 9:


T3:Parasite culturing

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3Animate like the addition of 0.9% NaCl to the red solution (addition should happen as in slide:11) and Animate the centrifugation step like in previous steps (slide:7)After the centrifugation step, show like taking out the tube with red solution on top and show a small piece of pellet (mostly pink color) at the bottom of the tube. Now instruct user to discard red top solution and animate accordingly with the help of pipette.Draw a bottle labeled as supplemented media and the animator should animate like user taking the pipette, set it to 1ml, clicking on it, taking the media and adding the media to the pink color substance in the tube.

Add 0.9% NaCl to the pellet and centrifuge at 2400 rpm for 5min. The supernatant was discarded and the pellet must be washed with supplemented media.

Step 10:

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Incubator

Culture flask


Step 10:


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3Animator should draw a tube labeled as “RBC” (content should be red) and the bottle labeled as “supplemented media”

Animate: like the user, taking the pipette and setting it to 250ul and drawing 250ul of RBC solution and drawing 4.75ml of supplemented media using measuring cylinder sepearately and adding it to the small pinkish substances in the tube.

Now animate like transferring the above contents to the culture flask as shown in figure

Draw a instrument labeled as incubator 37’C and animate like user keeping the flask inside it

Draw a clock showing 4 hrs.

Add 0.25ml of RBC and 4.75 ml of supplemented media to the pellet and transfer to the 5ml culture flask and incubate at 37’ C for 4hours. Later proceed to centrifugation.


supplemented media “RBC”

Step 11:

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Centrifugerotor


Step 11: Audio Narration


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Instruct user to take out the sample from the culture flask into the tube for centrifuge. The animator should draw a centrifuge as shown in the figure. Animate in such a way that user clicks on open button to open centrifuge lid and keep the tube inside the rotor (with lots of holes) as shown. The animator should animate like the user should click on setting and set 2400 rpm, 5seconds ay 4’C temperature and click “enter” and animate like closing the lid and click “start”. Show a clock running for 5minutes. Once the 5 minutes is done the user should open the lid by clicking “open” and remove the column out.

Please include the buttons like enter, set, start, open in the centrifuge.

After 4hours of incubation, centrifuge the content for 5minutes at 2400rpm.


Step 12:


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3 After centrifuge, let user take out the tube. Zoom the tube to show red liquid layer on top of pellet. Animator should draw a bottle labeled as “sorbitol”Animate like the user removes red color solution from the tube as discard and to the left over pellet (white substance) add sorbitol by setting the pipette for 1ml. Show a clock running for 5 minutes

Add sorbitol to the pellet and keep it undisturbed for 5mins, helps in growth culturing to maintain all the cells at same stage.


sorbitol

Step 13:



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3 Animator should draw a bottle labeled “Media with 3%hemocrit”

Animate like addition of the solution to the sample by setting the pipette to take required volume depending on the pellet size and add to the sample. Keep the sample tube inside the candle jar, please re-draw the figure.

Draw a instrument labeled as incubator 37’C as shown slide: 9

Reconstitute the pellet in media containing 3% hematocrit and incubate at 37'C in candle jar. This step helps in culturing of the cells.


3% hematocrit

Step 14:


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3 Show the synchronized culture in ring stage ie (draw a cell inside the culture with ring like structure) and the user should take out a smear with help of spatula from the culture plate, transfer it on a glass slide, so that a smear should be made on the glass slide. Now allow user to add Field’s stains using the filler. The user should take the filler to add on the smear.Draw a bottle labeled as Field’s stain.

Repeat the steps from slide 17 to 22, IF NO synchronization.

Take a smear of the culture in the glass slide and stain using Field’s stain to check the ring stage of parasite. If the ring stage of parasite has come up nicely, user can processed to next step or must do the synchronization step again.

Repeat the synchronization step 4 times


Field’s stain

Step 16:


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3 Animator should draw a culture flask labeled as RPMI1640

Animate like pouring the above mentioned synchronised culture to the RPMI 1640 media.

Transfer the 30 ml of synchronised culture to the RPMI 1640 and allow to grow till 9% parasitema is reached.


Step 17:


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3Animator should draw a bottle labeled as saponin and PBS

Animate like user taking the pipette, set the value for 1ml to for addition of PBS and saponin to the above sample.

Show like incubation on ice and a clock for 5 min.

Add PBS of pH7.4 and 1% saponin to the pellet and incubate on ice for 5 min


Step 18:


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3 The cells are lysed and the parasites are released from it and the solution becomes colorless due to the lyses of cell

Animator should animate like the breakage of cell and the release of the brown rings to the outside and slowly show the solution becoming colorless in the culture falsk.


Step 19:

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Centrifugerotor


Step 19: Audio Narration


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The animator should draw a centrifuge as shown in the figure. Animate in such a way that user clicks on open to open it and keep the tube inside the rotor (with lots of holes) as shown. The animator should animate like the user should click on setting and set 16000 g, 1 minute and click “enter” and animate like closing the lid and click “start”. Show a clock running for 5minutes. Once the 5 minutes is done the user should open the lid by clicking “open” and remove the column out.

Please include the buttons like enter, set, start, open in the centrifuge.

Centrifuge the content to separate the solution into two phases.


Step 20:


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3Animate like after centrifugation the animator should show a bottle labeled as PBS

Animate like removing the liquid part in the tube (after centrifugation) and show a white substances at the bottom of the tubeAnimate like the user opening the PBS and adding the 1ml solution to the white substance in the tube using pipette by setting the pipette to 1000ul. Animate the centrifugation step

Remove the supernatant and add 1 ml PBS to the pellet and centrifuge at 16000 g for 1 min at 4 C and the step is repeated till the solution becomes colorless.


PBS

Step 21:


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3 Animator should draw fridge named as”-80 C” and the user should open the door and keep the pellet(white substance at the bottom of the tube) containing tube inside

Place the pellet at -80 C until protein extraction is done.


Step 22



T4: Reagent Preparation

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3Show a measuring balance. A hand action when clicked by user should ON the Instrument, pick paper from rack, place it on the balance so that balance reads 0.03g and the user should press ”0” on the balance to make the reading to “0.00”. This action need to be performed for each reagent weighing.


Step 22 : Audio Narration


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Show the weigh balance (as in previous slide) a spoon to take the reagents to weigh , bottle containing urea. The user should click on the bottles to measure 4.8 g urea of urea

Draw a bottle labeled as distilled water and measuring cylinder.

Animate like the user pouring 10ml to the cylinder (zoom and show the measurement) and then to the weighed urea and show like mixing them as in next slide

Show the bottle labeled as protease inhibitor

Animate like the user taking the pipette and set the value and addition of both urea and protease inhibitor to the tube taken out from -80C

Prepare 8M urea solution, add protease inhibitor to the solution to avoid protein degradation. The solution now need to be treated for sonication.

T4: Reagent Preparation

Step 23:

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Beaker

Magnetic bead

T4: Reagent preparation



Show magnetic stirrer instrument. Let user place the beaker on it. Display the beaker containing powder at bottom, liquid layer on top and a magnetic bead at the bottom. Instruct user to ON the instrument, let user control the speed nob and regulate it accordingly to control the mixing speed in the beaker. Animate powder getting into the solution.Show a turbid solution turning colorless Video File: Magnetic stirrer

Step 24: T5:Protein extraction

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Description of the action/ interactivity

Instruct user to place the tube on ice, with cap open. Show the sonicator instrument, place the tube such that the tip of sonicator rod touches the solution in the tube. Click on hand so that the user adjusts rod inside the tube Now display the screen of sonicator , to make the necessary setup with help of user interaction.user should click on the sonicator to proceed with sonication. Allow the user to set the parameters as cycles :6, time 5sec , amplitude 20% .

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec ,20% amplitude with 59 sec gap. Sonication help protein extraction by cell lysis.

Video File: Sonication

Step 25:


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3 Draw a vortex mixer as shown in figure and the user should keep the tube on it and press “start “ and show a clock running for 30 seconds

T5:Protein extraction

Step 26:


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Animate like the user opening the CHAPS bottle and taking the it ,adding it to the above sample

Show a clock running 20 mins and show mixing every 5 minutes as shown in slide 40

Draw a fridge named as “4 C” and animate like the user opening the fridge keeping the content inside.

Add CHAPS to the sonicated sample and keep for 20 mins with intermittent mixing

Centrifuge the content at 16000 g for 30min at 4’C and store both supernatant and pellet. Now the sample can be stored and is ready for further analysis. Please go through the future IDD for more information.

T5:Protein extraction

Animation area

INTRACTION 1: In slide-12: In case the user comes across the less RBC count in the blood.

Instruction: user to change the sample and see to it that the sample contains more RBC’s are used for processing.

INTRACTION 2: In slide-22: provide user the culture with unsynchronized cell and user proceeding with it.

Instruction: animate user facing the problem with cell lysis and protein extraction steps. Instruct user to check the synchronization step and proceed further. Instructions/ Working area

Credits

Name of the section/stage

Interactivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 5-7

Slide 8-14

Slide 15-29

Slide 30-32

Slide 33-35

Questionnaire:APPENDIX

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Question 1

Plasmodium vivax/Plasmodium Falsiparam causes

a)Filariab)Malariac)Fever and coldd)Headache

Answer:Malaria

Question 2

Parasetima is defined

a)Number parasites in one RBCb)Number of infected RBC /100 RBCsc)Number of infected WBC/100 WBCsd)Number of parasites/100RBCs

Answer:b)Number of infected RBC /100 RBCs


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Question 3

NaCl is added to the culture to

a)Make RBC to lyseb)Make the culture saltyc)Make the parasite to growd)Make cell to grow

Answer:a)Make RBC to lyse

Question 4

CPDA bag contains

a)Calcium Phosphorus Dextrose Adenineb)Calcium Phosphate Detergent Aldosec)Citrate phosphate Dextrose Adenined)Citrate Phosphate detergent Adenine

Answer:c)Citrate phosphate Dextrose Adenine


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Question 5:

CPDA bag is used for

a)Growing parasiteb)Collecting the bloodc)Storing the parasited)Growing the RBC

Answer:b)Collecting the blood

Links for further reading

APPENDIX 2

Chen JH, Chang YW, Yao CW et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.Proc Natl Acad Sci U S A2004, 7;101(49):17039-44.

Eymann C, Dreisbach A, Albrecht D. A comprehensive proteome map of growing Bacillus subtilis cells.

Proteomics. 2004 :2849-76.

Maldonado AM, Echevarría-Zomeño S, Jean-Baptiste S. et al. Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis. Proteomics 2008, 71(4):461-72.

2DE Tutorials by Angelika Görg : http://www.wzw.tum.de/blm/deg/ BOOKS Biochemistry by Stryer et al., 5th editionBiochemistry by A.L.Lehninger et al., 3rd editionBiochemistry by Voet & Voet, 3rd edition

Summary

APPENDIX 3

Plasmodium proteome study involves critical protein extraction with each step

has to be done with atmost care to get the proteins from the parasite. Proper

cell disruption by sonication and pellet washing ensures good quality yield of

protein that can be used for the proteome study.

Documents

Related LOs: Culture handling, Blood properties > Prior Viewing – IDD:6 Serum extraction, IDD:1 Bacterial extraction > Future Viewing –IDD: 11 Protein