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 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 Title of the concept *The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license 1

Learning objectives 1 After interacting with this learning object, the learner will be able to: Define steps involved in production and maintenance of parasite culture Identify the steps involved for maintaining parasite culture Summarize protein extraction from the plasmodium culture Interpret the results of the experiment. Assess the troubleshooting steps involved in the experiments. 2 3 4 5 2

1 Master Layout 2 3 4 5 Blood processing (Slide:5-7) Parasite processing (Slide:8-14) Parasite processing (Slide:15-29) 3 Reagent preparation (Slide:30-32) 4 Protein extraction (Slide:33-35) 5 Display a image from each of these steps, with user click. 3

1 Definitions and Keywords 2 3 4 5 1. 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. 2 3 4 5 4

Description of the action Step 1: 1 T1: Blood processing CPDA Bag 2 3 Audio Narration Description of the action Animator 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. 4 5 5

3 Step 2: 1 2 4 5 T1: Blood processing ‏ rotor Centrifuge Video File: Centrifuge 6

Description of the action Step 2: 1 T1: Blood processing ‏ RBC Description of the action Audio Narration 2 Animate 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. 3 4 5 7

Description of the action Step 3: 1 T2: Parasite processing 2 37’C -80’C Audio Narration ‏ Description of the action 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. 4 5 8

3 Step 4: 1 2 4 5 T2: Parasite processing Video File: balancing Audio Narration (if any)‏ Description of the action 4 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. 5 Video File: balancing 9

Description of the action Step 5 : 1 T2: Parasite processing 2 NaCl 3 Description of the action Audio Narration ‏ Show 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. 4 5 10

Description of the action Step 6: 1 T2: Parasite processing 2 Description of the action Audio Narration ‏ Animator 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 3 4 5 11

Description of the action Step 7: 1 T2: Parasite processing 2 Audio Narration Description of the action 3 RBC are lysed and the parasites are released. RBC lyses results in the red color of the solution. 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. 4 5 12

3 Step 8: 1 2 4 5 T2: Parasite processing Audio Narration (if any)‏ Description of the action 4 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. 5 13

Description of the action Step 8 : 1 T2: Parasite processing 2 NaCl 3 Description of the action Audio Narration ‏ Let 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 NaCl Animate 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. 4 5 14

Description of the action Step 9: T3:Parasite culturing 1 2 Description of the action Audio Narration Animate 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. 3 4 5 15

Step 10: T3:Parasite culturing 1 2 3 4 Culture flask Incubator 5 16

Description of the action Step 10: T3:Parasite culturing 1 2 Description of the action Audio Narration supplemented media “RBC” Animator 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. 3 4 5 17

Step 11: T3:Parasite culturing 1 2 3 4 rotor Centrifuge 5 18

Description of the action Audio Narration Description of the action Step 11: T3:Parasite culturing 1 After 4hours of incubation, centrifuge the content for 5minutes at 2400rpm. 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. 2 3 4 5 19

Description of the action T3:Parasite culturing Step 12: 1 2 sorbitol Audio Narration Description of the action 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. 4 5 20

3 Step 13: 1 2 4 5 T3:Parasite culturing 3% hematocrit Audio Narration (if any)‏ Description of the action 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. 4 5 21

Description of the action Step 14: T3:Parasite culturing 1 2 Audio Narration Description of the action Field’s stain 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 4 5 22

Description of the action Step 16: T3:Parasite culturing 1 2 Audio Narration Description of the action 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. 4 5 23

Description of the action Step 17: T3:Parasite culturing 1 2 3 Audio Narration Description of the action Add PBS of pH7.4 and 1% saponin to the pellet and incubate on ice for 5 min Animator 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. 4 5 24

Description of the action Step 18: T3:Parasite culturing 1 2 Audio Narration Description of the action 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. 4 5 25

Step 19: T3:Parasite culturing 1 2 3 4 rotor Centrifuge 5 26

Description of the action Audio Narration Description of the action Step 19: T3:Parasite culturing 1 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. 2 3 4 5 27

Description of the action Step 20: T3:Parasite culturing 1 PBS 2 Audio Narration Description of the action Animate 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 tube Animate 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 3 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. 4 5 28

Description of the action Step 21: T3:Parasite culturing 1 2 Audio Narration Description of the action 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. 4 5 29

3 Step 22 1 2 4 5 T4: Reagent Preparation Audio Narration (if any)‏ Description of the action 4 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. 5 30

Description of the action Step 22 : 1 Audio Narration ‏ Description of the action T4: Reagent Preparation 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. 2 3 4 5 31

3 Step 23: 1 2 4 5 T4: Reagent preparation Beaker Magnetic bead Description of the action Audio Narration (if any)‏ 4 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 5 32

3 Step 24: 1 2 4 5 T5:Protein extraction Video File: Sonication Audio Narration (if any)‏ 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% . 3 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. 4 Video File: Sonication 5 33

Description of the action Step 25: 1 T5:Protein extraction 2 Audio Narration Description of the action 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 4 5 34

Description of the action Step 26: 1 T5:Protein extraction 2 Audio Narration Description of the action 3 Animator should draw a bottle labeled as CHAPS 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. 4 5 35

Instructions/ Working area Slide 8-14 Slide 5-7 Slide 15-29 Slide 30-32 Slide 33-35 Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07 Name of the section/stage 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. Interactivity area Button 01 Button 02 Button 03 Instructions/ Working area Credits 36

Questionnaire: APPENDIX 1 Question 1 Plasmodium vivax/Plasmodium Falsiparam causes Filaria Malaria Fever and cold Headache Answer:Malaria Question 2 Parasetima is defined a)Number parasites in one RBC b)Number of infected RBC /100 RBCs c)Number of infected WBC/100 WBCs d)Number of parasites/100RBCs Answer:b)Number of infected RBC /100 RBCs 37

Questionnaire: APPENDIX 1 Question 3 NaCl is added to the culture to a)Make RBC to lyse b)Make the culture salty c)Make the parasite to grow d)Make cell to grow Answer:a)Make RBC to lyse Question 4 CPDA bag contains a)Calcium Phosphorus Dextrose Adenine b)Calcium Phosphate Detergent Aldose c)Citrate phosphate Dextrose Adenine d)Citrate Phosphate detergent Adenine Answer:c)Citrate phosphate Dextrose Adenine 38

Questionnaire: APPENDIX 1 Question 5: CPDA bag is used for a)Growing parasite b)Collecting the blood c)Storing the parasite d)Growing the RBC Answer:b)Collecting the blood 39

Links for further reading APPENDIX 2 Links for further reading  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 edition Biochemistry by A.L.Lehninger et al., 3rd edition Biochemistry by Voet & Voet, 3rd edition 40

APPENDIX 3 Summary 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. 41