1.  Related LOs: Sonication > Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-6. Extraction of serum protein. > Future Viewing – IDD-11. Protein quantification, IDD-14. Isoelectric focusing, IDD- 17. SDS-PAGE  Course Name: Sonication on bacteria and serum protein extract  Level(UG/PG): UG  Author(s): Dinesh Raghu, Vinayak Pachapur  Mentor: Dr. Sanjeeva Srivastava Effect of Sonication on bacterial and Serum protein extraction Sonication is the process by which the high energy sound waves causing cell lyses, rupture of cell wall, causes release of contents from the cell. The method is more effective in cell disruption. Due to its high efficiency the method is highly suitable fro the protein extraction from the cell and the serum samples

2. Learning objectives The study helps the listener to learn : 1. Define the mechanism of cell lysing by sonication 2. Operate the steps involved in the extraction technique 3. Outline the precautions to be followed during the sonication. 4. Infer the steps involved to perform experiment. 5. Assess the troubleshooting steps involved in the experiments. 5 3 2 4 1

3. Master Layout 5 3 2 4 1 Serum Sample processing (Slide:4) Buffer Treatment (Slide: 5-6) Sonication (Slide: 7-10) Bacteria Sample processing (Slide:11-12 ) Buffer Treatment (Slide: 13) Sonication (Slide: 14-17)

4. Step 1: T1:Sample processing 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Animate removal of sample by opening the freezer and take the tube labeled as sample from -80 C and placing it on the ice for 15min, animate the effect with user interaction. During 15min zoom the tubes and animate the frozen solution changing to liquid phase. Show the user taking the pipette out removal of required amount of serum and transfer it to the new tube. Place the old tube in freezer. This should happen with user click. Kindly redraw the figures Remove the serum from -80 C and allow it to thaw by keeping it on ice for 15 min. Transfer the required amount of the serum to the fresh, clean eppendorf tube for further sample processing. Transfer of sample must be a quick process.

5. Step 2: 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Show phosphate buffer bottle, instruct user to set the pipette to 500ul, pipette out the buffer as the user clicks on it and adding it to the tube labeled as serum for dilution. The user should click on the pipette for the action to be done. Kindly redraw the figures Dilute the serum 5 times using phosphate buffer of pH 7.4, which helps to maintain the buffer during the sonication step. T2: Buffer addition

6. Step 3: 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Instruct user to vortex the tube, hand animate by picking the tube and placing on top of rubber pad for vortex and clicking “ON” button. During vortex, animate the mixing of solution. The user should click on start button for vortex. kindly redraw the figures Vortex the sample to achieve uniform mixing Vortex the sample to achieve uniform mixing. T2: Buffer addition

7. Step 3: 5 2 1 4 3 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 touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice. Now display the screen of sonicator, to make the necessary setup like pulses, time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settings. 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 5sec, 20% amplitude with 5sec gap. Sonication carries out cell lysis which helps for protein extraction. T3:Sonication

8. Step 3: T3:Sonication 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis Zoom in a aggregation as in the figure from the tube. Show the sound waves hitting the those aggregation and causing separation/ splitting of aggreagated protein and the release of contents. Please redraw the figure sonication

9. Step 3: 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity After sonication, zoom the tube, instruct user to click on the tube for the contents display. Animate the user interaction. After sonication, the cells are lysed now serum contains high abundance proteins like, albumin, IgA, IgG, haptoglobin along with low abundance protein. High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins. After sonication steps the sample is taken for further purification process. T3:Sonication

10. Step 3: 5 2 1 4 3 Purification steps same as IDD-6. Extraction of serum protein from Slide : 17-32. For more information on the extraction steps go through future viewing IDD.

11. Step 1: T1: Sample preparation (bacterial) 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Inoculation same as in IDD-1. Extraction of bacterial protein. Instruct user to open the lid of centrifuge and rotor. Zoom in the rotor, 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. animate this in the display where user can increase and decrease the values of set parameters. Animate the clock for 30min. Kindly redraw the figures Transfer the bacterial culture into the clean centrifuge tube under aseptic condition. Centrifuge the culture for 10 min at 12000 rpm maintaining at 4’C

12. Step 2: T1: Sample preparation (bacterial) 5 2 1 4 3 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 portion (supernatant) completely into the discard, the action should take place only when the user clicks on the pipette and the tube. Kindly redraw the figures Remove the supernatant completely without disturbing the pellet, now take the pellet for further processing.

13. Step 2: T2: Buffer treatment 5 2 1 4 3 Audio Narration (if any)‏ Description of the action/ interactivity Show phosphate buffer bottle, instruct user to pipette 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.

14. Step 3: T3: Sonication 5 2 1 4 3 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 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. Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec,20% amplitude with 5 sec gap. Sonication carries out cell lysis which helps for protein extraction.

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

16. Step 3: T3: Sonication 5 2 1 4 3 Audio Narration (if any)‏ Description of the action/ interactivity Carry out the 3 centrifuge process as described earlier. The user should click on “ Start” for the centrifugation to on. Please redraw the figure Centrifuge the contents to remove the debris and collect the supernatant for further processing.

17. Step 3: 5 2 1 4 3 Extraction steps are same as IDD-1. Extraction of bacterial protein from Slide : 16-32 For more information on the purification steps go through future viewing IDD.

18. Animation area Interaction 1: slide-14: user carrying out sonication without cooling on ice bath, no gap between the bursts and proceeds further. Instructions: let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts. Interaction 2: slide-14: user carrying out sonication for longer time. Instructions: animate the tube getting hot and starts melting. Excessive sonication will lead to heating and ice packs helps down in cooling the sample. Instructions/ Working area Credits Name of the section/stage: BACTERIAL CULTURE Interactivity area Tab 02Tab 03Tab 04Tab 05Tab 06Tab 07 Button 01 Button 02 Button 03 Tab 01 Slide 4Slide 7- 10 Slide 5-6 Slide 11-12 Slide 14-17Slide 13

19. Questionnaire: APPENDIX 1 Question 1 Amplitude used for the sonication of serum is a)20% b)30% c)40% d)50% Answer: 20% What is the purpose of sonication in bacterial protein extraction? a)To mix the culture b)To denature the protein c)To denature the nucleotides d)To lyse the cells Answer: To lyse the cells

20. Questionnaire: APPENDIX 1 What is the purpose of sonication in serum protein extraction? a)To mix the culture b)To deplete high abundance protein c)To denature the nucleotides d)To break the protein aggregates Answer: To break the protein aggregates

21. 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.Chen JHChang YWYao CW Eymann CEymann C, Dreisbach A, Albrecht D. A comprehensive proteome map of growing Bacillus subtilis cells.Dreisbach AAlbrecht D 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., 5 th edition Biochemistry by A.L.Lehninger et al., 3 rd edition Biochemistry by Voet & Voet, 3 rd edition

22. Summary APPENDIX 3 Ultrasonic waves generated by a sonicator lyse cells through shear forces. Complete shearing is obtained when maximal agitation is achieved, but care must be taken to minimize heating and foaming. Sonicate cell suspension in short bursts to avoid heating.Cool on ice between bursts. Sonication is a vigorous way of cell lysis.