Methodology for the extraction of Bacterial protein Extraction of the entire protein from the sample requires a optimized protocol to increase the protein amount in the extract. The protein extraction from the cell requires suitable reagents and technique that can yield a better and efficient result ‏ Related LOs: Cell culture, handling laminar air flow > Prior Viewing – IDD:2 Plant extraction, IDD:6 Serum extraction > Future Viewing – IDD:11 Protein Quantification, IDD: 12 Rehydration, IDD: 15 IEF, IDD:17 SDS-PAGE, IDD:20 Staining Course Name: Methodology for the extraction of Bacterial protein 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

Learning objectives 1 After interacting with this learning object, the learner will be able to: Identify the mechanism in TRIzol extraction Examine the technique involved in culture growth Interpret the results of the experiment Assess the troubleshooting steps involved in the experiments 2 3 4 5

1 Master Layout 2 3 4 5 Inoculation of Bacterial culture (Slide 5-8) Centrifugation followed by Buffer treatment (Slide 9-11) 2 Cell Lysis by sonication (Slide 12-14) Trizol treatment (Slide 12-14) 3 Chloroform treatment (Slide 19) Absolute alcohol treatment (Slide 20-21) Acetone precipitation (Slide 22-23) 4 Sample treatment with rehydration buffer (Slide 24-27) Store the sample at -20’C (Slide 28) 5

Definitions and Keywords 1 1.Protein: are the biomolecules, composing of amino acid, which forms the building block of the system and performs most of the function in the system. 2.Bacterial protein extraction: The process by the proteins from the cell are recovered for analysis purpose is called protein extraction. The chemicals involved in the extraction are a)Luria –Bertani broth (LB): The LB broth consists of yeast extract as carbon source, peptone as amino acid source, NaCl to maintain osmo-regulation and water. b)Trizol reagent: The reagent consists of phenol, guanidium thiocyanate and chloroform. Phenol and chloroform helps in phase separation while guanidium thiocyanate acts as Rnases inhibitors. c)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 zwitterioinc detergent and a constituent of rehydration buffer that is used to solubilize the proteins including membrane proteins. d) Urea: It is a organic compound in rehydration buffer that is used to denature protein. 2 3 4 5

3 Step 1: 1 2 4 5 T1:Inoculation of Bacterial culture Video File: LAMINAR air flow 3 Audio Narration (if any)‏ Description of the action/ interactivity Show a person sitting in front of laminar hood, zoom the laminar working bench having , tissue roll, ethanol bottle, burner, beaker with tooth picks. Instruct user to clean the bench. Allow user to pick tissue, wet it with ethanol and clean the whole working bench with user control like user should click on the hand so that the cleaning must happen. Now animate to close the laminar hood, click for “UV light” ON for 5min display the hood in blue color. Later after 5min, Click for “Light” “Blower” ON buttons to start the laminar hood. Please redraw the figures Clean the laminar unit thoroughly with ethanol and expose it to UV for further sterilization to avoid contamination. Later after 5min of UV exposure the laminar hood is ready for carrying out experiment. 4 5

3 Step 1: 1 2 4 5 T1:Inoculation of Bacterial culture LB Broth Audio Narration (if any)‏ Description of the action/ interactivity Take out a plate labeled as master culture plate from fridge, LB broth from the instrument labeled as incubator, the user should click on the hand to open the incubator, take the tube and close it and transfer it on laminar bench. With user control, open the lid of the culture plate, show few white dots in the plates as shown and the user should click on the hand to take a tooth pick and pick a colony from the master culture, close the culture plate. Take the “LB broth” tube unplug the cotton roll with left hand. The user should click on hand to remove the cotton plug. Now show like putting the tooth pick inside the LB broth, and contacting the solution with the tooth pick. Please redraw the figure. Pick a bacterial colony using the tooth pick from the master culture. Inoculate the bacterial colony to the sterile broth. Always keep the burner on during inoculation and perform the action close to burner to avoid any contamination. 4 5

Description of the action/ interactivity Step 1: 1 T1:Inoculation of Bacterial culture 2 3 Audio Narration Description of the action/ interactivity Show the placing of inoculated tube in incubator shaker. And the user should, open the lid, place the tube and close the lid. Instruct user to click on settings to set the temperature to 37’C and RPM 200 and start button so that inside stuff in the shaker starts moving horizontally. Please redraw the figures. Show a clock running for 8 hours Place the inoculated tube in the shaker-incubator at 37’C for 6-8 hours. 4 5

3 Step 1: 1 2 4 5 T1:Inoculation of Bacterial culture Audio Narration (if any)‏ Description of the action/ interactivity Once the time is complete instruct the user to take out the inoculated tube, open it. zoom in to show the yellow liquid turned to turbid. Show the transfer of grown culture into the clean centrifuge tube as the user clicks on it . This should happen inside the laminar hood as shown in slide 5 Please redraw the figure. Transfer the bacterial culture into the clean centrifuge tube under aseptic condition for further processing. 4 5

3 Step 2: 1 2 4 5 T2: Centrifugation followed by Buffer treatment . Audio Narration (if any)‏ Description of the action/ interactivity Instruct user to open the lid of centrifuge and drum. Zoom in the rotor, balance equal number of tubes inside the drum. Close the lid of rotor and of centrifuge with hand action. Instruct user to set the rpm (12000), temperature(4’C) and time (10min) parameters, along with display. User can increase and decrease the values of set parameters. Animate the clock for 30min. Kindly redraw the figures. Centrifuge the culture for 10 min at 12000 rpm maintaining at 4’C to harvest/collect the culture. . 4 5

3 Step 2: 1 2 4 5 T2: Centrifugation followed by Buffer treatment . Audio Narration (if any)‏ Description of the action/ interactivity After 10min, instruct user to open the lid of centrifuge, rotor and animate the hand action to left the tube from rotor. Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure. Now pipette out top liquid portion (supernatant) completely and pipette into empty tube, the action should take place only when the user clicks on the pipette and tube. Kindly redraw the figures. Remove the supernatant completely without disturbing the pellet, now take the pellet for further processing. . 4 5 Video File: Centrifuge.MTS and Centrifuge_part2.MTSc

3 Step 2: 1 2 4 5 T2: Centrifugation followed by Buffer treatment . Audio Narration (if any)‏ Description of the action/ interactivity Show phosphate buffer bottle, instruct user to set the pipette to 1000ul and take the buffer in the pipette and transfer it into the tube. The user should click on the pipette for the action to be done. Kindly redraw the figures Wash the pellet with phosphate buffer thoroughly to remove the excess broth. Once broth is removed completely, cell lysis need to be carried out. . 4 5

3 Step 3: 1 2 4 5 T3: Cell Lysis by 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 now the user should click on the instrument to adjust the rod to dip into the solution. Now display the screen of sonicator, to make the necessary set up with help of user interaction like setting in the amplitude, time and pulses as in right hand side. user should click on the sonicator to proceed with sonication. Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec, 20% amplitude with 5 sec gap. Sonication helps protein extraction by cell lysis. 4 5 Video File: Sonication

3 Step 3: 1 2 4 5 T3: Cell Lysis by sonication High frequency sound waves 2 3 Audio Narration (if any)‏ Description of the action/ interactivity Zoom in a cell from the tube. Show the sound waves hitting the cell and causing cell lyses and the release of contents. Please redraw the figure High frequency sound waves break open the cell wall and the contents are released into the buffer . 4 5

3 Step 3: 1 2 4 5 T3: Cell Lysis by sonication . Audio Narration (if any)‏ Description of the action/ interactivity Carry out the 3 centrifuge process as described in slide 9. The user should click on “ Start” button for the centrifugation to ON. Please redraw the figure. Now show the tube containing two layers (liquid and white substance and ask user to set the pipette and take the liquid part to fresh tube. Centrifuge the contents to remove the debris and collect the supernatant for further processing. . 4 5

3 Step 4: 1 2 4 5 T4: Trizol treatment Guanidium thiocyanate Phenol Audio Narration (if any)‏ Description of the action/ interactivity The user should click on the trizol to know the constituents of trizol reagent. please redraw the figure Trizol reagent consists of guanidium thiocyanate, phenol,chloroform that separates DNA, RNA and proteins from each other within the solution. 4 5

3 Step 4: 1 2 4 5 T4: Trizol treatment Audio Narration (if any)‏ Description of the action/ interactivity The user must click on the pipette, set it 1000ul and take the trizol so that the trizol is added to the supernatant(liquid in fresh tube). And the user should click on vortex to start show like the content is mixed and color should be pink. Please redraw the figures. Add trizol to the supernatant and vortex it thoroughly. 4 5 Video File: Vortex

3 Step 4: 1 2 4 5 T4: Trizol treatment Chloroform Audio Narration (if any)‏ Description of the action/ interactivity Show the bottle labeled as chloroform and the user should click on the bottle for addition of chloroform to the sample when the user clicks on the pipette, set to 200ul and take the reagent and add to the liquid part. Place the tube over the vortex. And show the mixing, solution should be milky in appearance. Please redraw the figure. Add chloroform to the sample, mix thoroughly till the color appear like milk-shake and keep at room temperature for 5min till you get the phase separation. 4 5

3 Step 4: 1 2 4 5 T4: Trizol treatment RNA Protein DNA Audio Narration (if any)‏ Description of the action/ interactivity Carry out centrifuge step as discussed earlier (slide:9) after centrifuge zoom the tube to show formation of three layers like in figure. Please redraw the figure. User should click on the layer to know their constituents. Three layers are formed after centrifugation. The top layer containing RNA, Middle layer with protein and the bottom layer with DNA. 4 5

3 Step 5: 1 2 4 5 T5: Absolute alcohol treatment Audio Narration (if any)‏ Description of the action/ interactivity Show the removal of RNA (top) layer for discard, when the user clicks on the pipette. Show the bottle labeled as alcohol and the user should set the pipette to 300ul and take the alcohol in pipette and add to the tube and mix well by inverting the tubes with hand. The user should click on the pipette so that the action takes place. Later carry out centrifugation step as in slide:9. Please redraw the figure. Remove the aqueous layer containing RNA without disturbing the other two layers. RNA can be stored or discarded depending on the requirements. Add the absolute alcohol to the remaining layers and mix gently till the middle layer dissolve and keep at room temperature for 3 min. Centrifuge the content for 5 min at 2000 rpm. 4 5

3 Step 5: 1 2 4 5 T6: Acetone treatment Supernatant containing Protein DNA 2 3 Audio Narration (if any)‏ Description of the action/ interactivity Zoom the tube after centrifuge, to show two layers and user click on each layer to know about it. Show the bottle labeled as acetone and the user should set the pipette to 1000ul and take the acetone in pipette and add to the tube and mix well with vortex as described earlier. Animate addition of acetone to the sample when the user clicks on the pipette and vortex should be done when the user clicks on the ON button. Please redraw the figure DNA forms pellet and the supernatant containing protein is recovered. Add chilled acetone to the sample and vortex it, this helps to precipitates the protein. 4 5

3 Step 5: 1 2 4 5 T6: Acetone treatment Audio Narration (if any)‏ Description of the action/ interactivity Show placing of sample at -20 C for 1 hr by opening the freezer. The user should click freezer to open it, keep the sample and close it. Show the clock running for an hour. Instruct user to take out the sample to carry out centrifuge step. centrifugation as in slide:9. After centrifuge zoom the tube to show white substance (pellet) formation. Please redraw the figures. Place the sample at -20 C for at-least an hour for complete precipitation. Centrifuge the content to get white pellet to be seen at the bottom. 4 5

3 Step 6: 1 2 4 5 T7:wash buffer preparation Audio Narration (if any)‏ Description of the action Audio Narration (if any)‏ Show a measuring balance, with display, ON, OFF and TARE/0 buttons on it. let user ON it, display reading as 0.000g, let user picks up the paper from the rack, makes 1/10 of folding on the sides and places it on the balance. Now the display reading changes to 0.003g. Instruct user to TARE the reading. And animate to click the tare button. Once user clicks it, reading must show ”0.000” During the material measurement on paper, the weight of the paper need to be tarred or set to zero before weighing. 4 5 Video File: Measuring Balance 22 22

Description of the action Step 6: 1 T7:wash buffer preparation Guanidine-HCL 2 ethanol Description of the action Audio Narration 3 Let user pick up guanidine HCl with spatula, measuring cylinder from the rack and place it on the table next to balance. Instruct user to weigh 0.35g of guanidine let user tare the balance, user should click on the guanidine bottle, uncap it, with help of spatula weigh the required amount on a paper over the balance. Display a gradual increase in reading with quantity addition. if the gram exceeds user should remove some quantity or if it less add the quantity to get the exact required amount. After weighing transfer the quantity to beaker. Now click on ethanol and the user should pour in measuring cylinder till volume reaches 10ml and add to the beaker containing weighed guanidine and show like mixing well in the vortex as shown earlier. Prepare wash solution, washing is carried to remove excess broth and other contaminants if present in the solution. 4 5 23 23

Description of the action Step 6 : 1 T7: buffer preparation CHAPS 2 Rehydration buffer (RB) water urea 3 Audio Narration Description of the action Instruct user to prepare rehydration buffer (RB). Animator redraw above figure as shown. Let user takes out the bottles from the racks. Instruct user to weigh CHAPS and Urea, tare the balance like in slide:22, the user should pick the spatula open the lid of the CHAPS to weigh 0.02g and Urea bottle to weigh 0.6g separately and put in the tube labeled as RB. Instruct user to click on water bottle, take out 1ml pipette, set it for 1000ul and pipette in water into RB bottle. let user take the bottle for a brief vortex to mix the solution. Weigh 0.6g of urea, 0.02g of CHAPS to prepare rehydration buffer which must be store at 4’C. 4 5 24

3 Step 7: 1 2 4 5 T8: Pellet washing Audio Narration (if any)‏ Description of the action/ interactivity Show the removal of supernatant as the user clicks on pipette, only liquid part has to be removed ,show like opening the tube and keeping the pellet in room temperature show like drying and pellet drying need to animated. Show the reagents which constitutes the washing solution. The user should click on the chemical to get their role. Show like the user taking the pipette set to 1000ul and take the reagents and add to the tube And carry out centrifuge as in slide 9. Show like repeating the step for 4 times. Remove the liquid part in each step and add the washing solution till protein become white. Kindly redraw the figures Remove the supernatant carefully and air dry the pellet. Pellet retains the pink color, carry out washing the pellet with 0.3 M guanidium –HCl in 95% ethanol for 4 times to remove the color and for inactivation of RNAses. Each pellet wash need to be followed by centrifugation by discarding the supernatant till pellet becomes color-less. 4 5

3 Step 8: 1 2 4 5 T8: Rehydration buffer treatment Urea CHAPS 2 3 Audio Narration (if any)‏ Description of the action/ interactivity Zoom-in tube showing the colorless pellet. Instruct user to add rehydration buffer by setting the pipette to 400ul and take the solution and add to it. Display the contents of rehydration buffer when user clicks on the tube. Kindly redraw the image The rehydration buffer consists of CHAPS used to solubilize the proteins including membrane proteins. Urea used to denature protein structure. 4 5

3 Step 7: 1 2 4 5 T8: Rehydration buffer treatment Audio Narration (if any)‏ Description of the action/ interactivity Instruct user to add rehydration buffer to sample with pipette and animate the step accordingly. After addition close the cap of tube and place it on rubber pad of the vortex for proper mixing. Animate the pellet get disappearing into the solution. kindly redraw the images. Add 0.4 ml of rehydration buffer (IDD-2 Extraction of serum protein, slide 8) to the dried pellet and vortex till the pellet completely goes into the solution. 4 5

3 Step 8: 1 2 4 5 T8: Sample storage at -20’C Audio Narration (if any)‏ Description of the action/ interactivity Zoom-in tube with clear solution, instruct user to store the tubes at -20’C into the freezer . Animate opening the door of freezer, placing the tube and closing the door. kindly redraw the images. The sample in rehydration buffer can be stored at -20’C and can be thawed and used during sample Quantification. Please go through the future viewing IDD for more information. 4 5

Instructions/ Working area Slide 9-11 Slide 19 Slide 12-14 Slide 12-14 Slide 20,21 Slide 5-8 Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Animation area INTERACTION 1: In Slide-9: let user place the centrifuge tube in rotor without balancing and proceeds with the setup. Instruction: Display on monitor of a centrifuge error message saying “Improper balancing” and let user stop the centrifuge, open the lid and of the rotor to place the tube with same volume in each tubes with balanced across the rotor and start the setup again. Interactivity area Button 01 Button 02 Button 03 Instructions/ Working area Credits

Instructions/ Working area Slide 22,23 Slide 24-27 Tab 07 Tab 08 Tab 09 Tab 10 Tab 05 Tab 06 Tab 07 Name of the section/stage Animation area Interactivity area Button 01 Button 02 Button 03 Instructions/ Working area Credits

Questionnaire: APPENDIX 1 Question 1 What is the amino acid source in LB broth? Glucose Amino acid Peptone Nacl Answer : Peptone Question 2 What is the purpose of sonication? To mix the culture To denature the protein To denature the nucleotides To lyse the cells Answer: To lyse the cells

Questionnaire: APPENDIX 1 Question 3 The reagent that separates the proteins and DNA-RNA from the cell is Phenol Glucose Rehydration buffer Trizol Answer : Trizol Question 4 What is the constituent(s) of the upper layer after trizol separation? RNA DNA and RNA DNA,RNA and proteins Proteins and DNA Answer: RNA

Questionnaire: APPENDIX 1 Question 5 The chemical that inactivates the RNases is a) Trizol b)Phenol c)Guanidium –Hcl d)Nacl Answer : Guanidium -Hcl

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

APPENDIX 3 Summary The protein extracted should be devoid of the phenolics from the trizol reagent which may hinder separation in IEF and SDS. 95%ethanol wash will remove such compounds from the sample Sonication is more important step as it disrupts the cell which makes the proteins to come out of the cell. Care should be taken to prevent the unwanted particles in the extraction.