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Methodology for the extraction of Brain tissue protein

Methodology for the extraction of Brain tissue protein.

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Methodology for the extraction of Brain tissue protein

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  1. Methodology for the extraction of Brain tissue 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 from different samples. The method explained here mainly focuses on the effective method of extracting protein from the brain tissue ‏ • Related LOs: Liquid nitrogen properties, using mortar and pestle > Prior Viewing – IDD:1 Bacterial extraction, IDD:2 Plant protein extraction > Future Viewing – IDD: 11 Protein quantification, IDD: 14 Isoelectric focusing, IDD: 17 SDS-PAGE, IDD: 20 Staining, IDD: 24 Data analysis • Course Name: Extraction of brain tissue protein • Level(UG/PG): UG • Author(s) : Dinesh Raghu, Vinayak Pachapur • Mentor: Dr. Sanjeeva Srivastava

  2. Learning objectives 1 After interacting with this learning object, the learner will be able to: Define protein extraction of brain tissue using lysis buffer and homogenisation. Identify protein solubilisation using rehydration buffer. Operate steps involved in handling the instrument and the materials used. Interpret the results of the experiment. Assess the troubleshooting steps involved in the experiments. 2 3 4 5

  3. Master Layout 1 Weighing the sample (Slide:6-7) Liquid Nitrogen treatment (Slide:8-10) 2 Grinding the Brain tissues (Slide:11) Lysis Buffer treatment (Slide:12-14) 3 Tissue homogenization (Slide:15-16) Protein precipitation at -20’C (Slide:17) Centrifugation and Acetone treatment (Slide:18-22) 4 Rehydration buffer treatment (Slide:23-24) • Sample storage at -20’C (Slide:25) 5 Animate for user click to show images or instruments used for each step from the respective slides.

  4. Definitions and Keywords 1 • 1. Protein : Proteins are the biomolecules, composed of amino acid, forming the building block of the system and performs most of the biological functions of the system. • 2. Protein extraction: The process by the proteins from the cell are recovered for the analysis purpose is called protein extraction. The chemicals involved in the extraction are • 3. Lysis Buffer: A cocktail of reagents used for cell lyses. • a) Trichloro acetic acid: The acid used in the lyses buffer for lyses of the cell and helps to precipitate the protein. • b) Acetone: One of the constituent of lyses buffer used to denature the protein. • c) Dithiothreitol: Constituent of lyses buffer used to reduce the disulfide bonds in the protein. • 4. Rehydration buffer: A cocktail of reagents used for sample solubalization and used for sample storage. • a) CHAPS: is a zwitter-ionic detergent, constituent of rehydration buffer that is used to solubilize the proteins including membrane proteins. • b) Urea: It is a organic compound in rehydration buffer that is used to denature protein. • c)Bromophenol blue: Used in rehydration as color marker and acid –base indicator 2 3 4 5

  5. Definitions and Keywords 1 5. SB-14 Sulfobetaine , a Zwitterionic detergent used for the protein solubilization , it maintains the zwitterionic property in wide range of pH. 2 3 4 5

  6. Step 1: T1: Weighing the sample 1 2 3 Description of the action Audio Narration (if any)‏ Show a measuring balance the user should click ON the Instrument, pick tube from rack, fold it across and on the edge, 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”. Animate the action, whenever user starts to weigh any reagents. Clean the surface of the balance, Tare the weight of the paper before weighing for each reagent. 4 5 Video File: Balancing

  7. Weighing balance Description of the action/ interactivity Audio Narration (if any)‏ First show the brain tissue in a small tube. Transfer the brain tissue, with forceps on paper for weighing as shown in figure. show the picking of the tissue with forceps and dropping into the eppendorf tube in the measuring balance should show the change in the measurement till it reaches 50 mg. each time the user should click on the hand for the event to happen. Step 1: T1:Weighing the sample 1 2 3 The tissue were stored at -80C and kept in ice during weighing. Weigh 50mg of cleaned brain tissue for the protein extraction. 4 5

  8. Description of the action/ interactivity Audio Narration (if any)‏ • Animate to click on liquid nitrogen bottle, lift it to pour slowly on pestle and mortar. Change the appearance of pestle and mortar to frozen phase to show a pre chill effect. when the user clicks on the liquid nitrogen should pour into the pestle . Kindly redraw the figures from Look out for IDD:2 Extraction of plant protein, slide:12. Step 2: T2:Liquid Nitrogen treatment 1 2 Liquid nitrogen 3 Add liquid nitrogen to mortar and pestle to prechill them. Care should be taken while pouring not to spill out and action to be carried out away from body. To bring down the temperature of mortar and pestle, this step is carried out. 4 5

  9. Description of the action/ interactivity Audio Narration (if any)‏ First show the pre-chilled mortar and pestle, transfer the weighed brain tissue into the mortar. Animate user transferring brain tissue with forceps and dropping into the pre-chilled mortar. Please redraw the figure for brain tissue into the chilled pestle. Step 2: T2:Liquid Nitrogen treatment 1 2 3 Transfer the 50mg of Brain tissue sample from eppendorf tube into the pre-chilled mortar 4 5

  10. Description of the action/ interactivity Audio Narration (if any)‏ Show the tissue in pre-chill mortar , liquid nitrogen bottle. instruct user to add liquid nitrogen. when the user clicks on the liquid nitrogen it should pour into the mortar. Animate the tissue changing to freeze crisp ones. Kindly redraw the figures Step 2: T2:Liquid Nitrogen treatment 1 2 3 Add 10ml of liquid nitrogen to the tissue, add it at one go. After addition of nitrogen a crisping noise is heard. 4 5

  11. Description of the action/ interactivity Audio Narration (if any)‏ Show the hand moment like grinding on tissue sample, grinding should be carried only when the user continuously clicks on the pestle image and it should stop when the user unclick on it. While grinding animate the large tissue, to small pieces later turning into powder form. Kindly redraw the figures Step 3: T3:Grinding the Tissue 1 2 3 Grind the sample until it looks powdery. 4 5

  12. Description of the action/ interactivity Audio Narration (if any)‏ Zoom in lyses buffer tube, animate like unscrewing the cap, pipette in with hand action setting to 1ml in the pipette, taking the lyses buffer and add on powdered sample in mortar. Kindly redraw the figures. Event should happen only when the user clicks on the pipette. Step 4: T4:Lysis Buffer treatment 1 2 Lysis Buffer 3 To the powdered sample add the lysis buffer (Buffer preparation in IDD:2 Plant extraction, slide:16) 4 5

  13. Trichloroacetic acid Acetone Dithiothreitol Description of the action/ interactivity Audio Narration (if any)‏ Animate grinding effect with display with explanation of lysis buffer composition with structure. Kindly redraw the figures Step 4: T4:Lysis Buffer treatment 1 2 3 The lysis buffer consists of trichloroacetic acid aids in lysis of the cell and precipitate the protein, acetone used to denature the protein and dithiothreitol used to reduce the disulfide bonds in the protein. 4 5

  14. Description of the action/ interactivity Audio Narration (if any)‏ Show the grinding of the sample and making into paste ,the user should click continuously on pestle so that the grinding will be carried out. Kindly redraw the figures for tissue sample IDD: 2 Plant extraction, slide:11. The paste must be pink in color , please redraw the figure. Step 4: T4:Lysis Buffer treatment 1 2 3 The powdered sample should be grounded until it becomes paste. 4 5

  15. Step 5: T5:Tissue homogenization 1 2 3 Description of the action/ interactivity Audio Narration (if any)‏ Animate like the user taking the pipette and setting the value to 1000ul and pipetting out the lyses buffer when the user clicks on it and adding it to the paste (pink in color) and grinding again. Now show the transfer of the paste from the MP to the eppendorf tube, the transfer should be done, with user taking out the paste from the MP with spatula, and transferring it to tube. This should happen with user click. Kindly redraw the figures Transfer the paste to the clean, autoclaved eppendorf tubes for homogenization. 4 5

  16. Description of the action/ interactivity Audio Narration (if any)‏ Show homogenizer instrument, instruct user to pick the instrument and in other hand the eppendorf tube in ice bucket. Now dip the rod into the tissue solution as shown in the figure. On the instrument by clicking the ON button. Increase the levels accordingly. Animate the solution getting mixed in the tube. Kindly redraw the figures Step 5: T5:Tissue homogenization 1 ON Button 2 3 Homogenize the tissue so that the cell lyse and the contents are released. Initially begin with low speed, later increase the speed accordingly. Homogenization is done by placing the tube in ice 4 Video File: Homogenizer 5

  17. Description of the action/ interactivity Audio Narration (if any)‏ Animate the hand picking the ependroff tubes and placing them at -20 C incubator by opening the incubator. Once all the tubes are placed, close the door of incubator and animate the clock for 1hr. Kindly redraw the figures. Follow the further steps which are similar to IDD:2 Plant protein extraction, slide:18. Step 6: T6:Protein precipitation at -20’c 1 2 3 Place the sample at -20 C incubator for 1hr so that the protein precipitates 4 5

  18. Description of the action/ interactivity Audio Narration (if any)‏ After 1hr, instruct user to open door, take out the tubes, transfer into centrifuge. Zoom in the drum, balance equal number of tubes inside the drum of centrifuge . Close the lid of drum and of centrifuge with hand action. Instruct user to set the 14000 rpm, 4’ C temperature and 30 minutes time along with display. User can increase and decrease the values of set parameters. Animate the clock for 30min. Kindly redraw the figures Step 7: T7: Centrifugation and Acetone wash 1 2 3 Place the sample in the centrifuge, balance with equal number of tubes and centrifugation should be carried out at 4’C at 14000 rpm for 30 minutes for the separation of the phases. 4 5 Video File: Centrifuge

  19. Description of the action/ interactivity Audio Narration (if any)‏ After 15min, instruct user to open the lid of centrifuge, drum and animate the hand action to left the tube from drum. Now zoom the tube having two different solutions, bottom one opaque and top one transparent phase. Now pipette out top portion (supernatant) completely and discard into empty tube ,the action should take place only when the user clicks on the pipette and tube. Kindly redraw the figures Step 7: T7: Centrifugation and Acetone wash 1 2 3 After centrifuge, protein being heavier in nature settles down as pellet leaving out unwanted particles as supernatant. Remove as much as supernatant from the tube ,without disturbing the pellet and discard it . 4 5 Video File: Centrifuge

  20. Description of the action/ interactivity Audio Narration (if any)‏ Show tubes with pellets next to acetone bottle. Instruct user to add acetone and animate the step. action should be done only when the user clicks on the pipette. Kindly redraw the figures Step 7: T7: Centrifugation and Acetone wash 1 2 3 Add 1ml of ice cold acetone to the pellet for wash. 4 5

  21. Description of the action/ interactivity Audio Narration (if any)‏ Zoom in tube with pellet and acetone over it. Instruct user to vortex the tube, hand animate by picking the tubes and placing on top of rubber pad for vortex. During vortex, animate the pellet disappearing into the solution. The user should click on the hand and start the vortex so that the tube will be vortex. kindly redraw the figures and show the solution looks turbid. Step 7: T7: Centrifugation and Acetone wash 1 2 3 Vortex the pellet until it goes completely into the acetone. 4 5 Video File: Vortex

  22. Description of the action/ interactivity Audio Narration (if any)‏ Open lid of centrifuge and its drum. Zoom in the drum, balance equal number of tubes inside the drum of centrifuge . Close the lid of drum and of centrifuge with hand action. Instruct user to set the 14000 rpm, 4’C temperature and 5minutes time parameters, along with display. User can increase and decrease the values of set parameters. Animate the clock for 5min. Kindly redraw the figures Step 7: T7: Centrifugation and Acetone wash 1 2 3 Discard the supernatant, If pellet retain color, centrifugation and acetone wash step should be done until the pellet becomes colorless. 4 5

  23. Urea CHAPS Description of the action/ interactivity Audio Narration (if any)‏ Zoom-in tube showing the colorless pellet. Instruct user to add rehydration buffer. Display the contents of rehydration buffer when user clicks on the tube. Kindly redraw the image. The event should happen when the user clicks on the pipette Step 8: T8: Rehydration buffer treatment 1 2 Sulfobetaine 3 The rehydration buffer consists of CHAPS and sulfobetaine used to solubilize the proteins including membrane proteins. Urea used to denature protein urea. 4 5

  24. Description of the action/ interactivity Audio Narration (if any)‏ Instruct the user to set 400ul in the pipette Instruct user to add 400ul 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. Step 8: T8: Rehydration buffer treatment 1 2 3 Add 400ul of rehydration buffer to the dried pellet and vortex till the pellet completely goes into the solution. 4 5

  25. Description of the action/ interactivity Audio Narration (if any)‏ 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. Step 9: T9: Sample storage at -20’C 1 2 3 The sample in rehydration buffer can be stored at -20’C and can be thawed and used during the experiment. The sample can be directly taken for protein quantification,1D, 2D run, can be cleaned up, do look out for the future viewing IDD for more information. 4 5

  26. Button 01 Button 02 Button 03 Slide 8 - 10 Slide 15 - 16 Slide 11 Slide 12 - 14 Slide 17 Slide 1 - 5 Slide 6-7 Introduction Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Animation area INTERACTIONS 1: slide-7: instruct user to weigh brain sample in range from 50, 100 and 150mg each for separate processing. Instructions: let user list out the problems faced during the processing and how user plans to carry out the step. INTERACTIONS 2: slide-16: while starting homogenization, user starts off with high speed and looses out the sample. Instructions: to check the volume in the sample, dip the rod accordingly, initially start at low speed and later the speed can be increased. Interactivity area Instructions/ Working area Credits

  27. Button 01 Button 02 Button 03 Slide 18-22 Slide 23 - 24 Slide 25 Tab 07 Tab 08 Tab 09 Tab 10 Tab 05 Tab 06 Tab 07 Name of the section/stage Animation area Interactivity area Instructions/ Working area Credits

  28. Question 1 The purpose of liquid nitrogen addition Maintain ice cold condition Makes the tissue brittle and easy for making it powdery To react with the tissue for easy extraction of the sample All the above Both a&b Answer: Both a&b Question 2 Homogenizer is for • Cell lyses • Tissue lyses • Mixing the reagents • Making a homogeneous solution Answer: Cell lyses Questionnaire: APPENDIX 1

  29. Questionnaire: Question 3 What is the purpose of incubating the lysed sample at -20 C for 1hr • Protein solubilization • Protein denaturation • Protein mixing • Protein Precipitation Answer: Protein Precipitation Question 4 What is the purpose of incubating the sample with rehydration buffer at 4C for overnight • Protein solubilization • Protein denaturation • Protein mixing • Protein Precipitation Answer: Protein Solubilization APPENDIX 1

  30. Questionnaire: APPENDIX 1 Question 5 The purpose of sulfobetaine addition in rehydration buffer • To increase the rehydration • To increase the protein denaturation • To increase the protein solubilization • To increase the protein separation property of IEF instrument Answer:To increase the protein solubilization

  31. 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

  32. APPENDIX 3 Summary The experiment mainly focuses on the extraction of protein from the brain tissue. As the sample taken for extraction is very less considering its low availability it is very important to get the maximum proteome from the lower quantity sample. So the extraction method discussed here shows maximum efficiency in extraction of the protein from the brain sample.

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