1. Isoelectric Focussing Proteins exhibit unique iso-electric property, such unique property of the proteins are exploited in the separation of individual proteins in a pool of proteome. Each protein can be separated from one another by iso-electric property ‏  Related LOs: IPG strips, > Prior Viewing –IDD-6. Extraction of serum protein, IDD-11. Protein quantification, IDD-13. Cyanine dye labeling > Future Viewing – IDD-16. Equilibration of IPG strips, IDD-17. SDS-PAGE, IDD-20. Silver staining  Course Name: Isoelectric Focussing  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

2. Learning objectives After interacting with this learning object, the learner will be able to: 1. Define first dimension separation of proteins using Iso-Electric Focusing (IEF) system. 2. Operate steps involved in handling the instrument and the materials used. 3. Recall the background to Iso-Electric Focusing (IEF). 4. Infer the steps involved to perform in the experiment. 5. Assess the troubleshooting steps involved in the experiments. 5 3 2 4 1

3. Master Layout 5 3 2 4 1 Clean and Dry the manifold (Slide:5) Position the manifold (Slide:6) Transfer IPG strips to manifold (Slide:7) Pour Cover fluid (Slide:9) Position Electrode assembly (Slide:10) Switch ON the instrument (Slide:11) Mechanism of separation (Slide:22-23) Completion of IEF (Slide:24) Storing the strip (Slide:25-26) Set program parameters and run IEF (Slide:12-21) Moisten and place the electrode wicks (Slide:8)

4. Definitions and Keywords 1. Protein: Protein are the biomolecules which is composed of amino acid,forms the building block of the system and performs most of the function of the system. 2. IEF: Isoelectric Focusing is an electrophoretic method that separates proteins according to their isoelectric points (pI). 3. IPG strip: An immobilized pH gradient strip is created by covalently incorporating a gradient of acidic and basic buffering groups (immobilines) into a polyacrylamide gel at the time it is cast. 4. Cover Fluid: Is used to minimize evaporation and thus prevent urea crystallization. 5. Paper Wicks: The wicks absorb excess water, salts, and proteins 6. Electrode assembly: Ensure good contact between the paper wick and IPGStrip gel for Iso electric focusing. 7. Manifold: is made of a thermally conductive aluminum oxide ceramic that rapidly dissipates heat to avoid potential “hot spots.” 5 3 2 4 1

5. Step 1: T1:Cleaning and Drying the IPG tray 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Draw manifold, a hand picture holding tissue paper. Instruct to click on hand, place it on the manifold to make a movements for cleaning. Draw manifold image from wet to final dry image. Place the manifold on the table. Take a clean tissue paper. Clean all the lanes of manifold, to make it free from dust and dry it completely.

6. Step 2: Audio Narration (if any) ‏ Description of the action/ interactivity T2: Position the manifold 5 2 1 4 3 User must perform opening the lid with a hand press on top of lid. Pick the manifold and position it in the instrument and level the manifold by placing the round spirit level on top of it with hand action. Place the instrument on level surface, Take the manifold and position it inside the groove of the instrument. Ensure that the surface is level by placing the round spirit level and adjust if necessary at the back edge of the instrument.

7. Step 3: 5 2 1 4 3 Pick the strip end with forceps from reswelling tray (which is kept for passive rehydration). Drain out excess oil by tapping it on tissue paper without folding the strip, keep it straight. Place the positive side of strip on the positive end of instrument with gel side up. T3: Transfer IPG strips to manifold Audio Narration (if any) ‏ Description of the action/ interactivity Mark “+” anode and “–” cathode end inside the instrument to differentiate. Show the hand action holding the forceps, picking the strip from the reswelling tray, tap it on the tissue paper for 30sec (show clock) and transfer to manifold. The + side of the strip has to be placed on + end. Make sure the strip is placed in the middle of the lane + -

8. Step 4: T4: Moisten and place the electrode wicks 5 2 1 4 3 Redraw the images with labels. Make user to hold the scissor and cut the wick with size (.25x1cm) into 2 pieces. User should be able to hold wick in one hand with forceps and with other hand pipette out 0.5ml distilled water on wick piece to make it wet. Place the wet wicks on each end of strips such that one end of the wick overlaps the end of the gel on the strip (zoom this part). Cut the paper wicks into 2 pieces. Add distilled water to the wicks until it gets wet. Place it on the strip end such one end of the wick overlaps the end of the gel on the strip. The paper wicks absorbs impurities /salts and aid in current conductance Audio Narration (if any) ‏ Description of the action/ interactivity wicks

9. Step 5: T5: Pour Cover fluid 5 2 1 4 3 Draw a bottle labeled as cover fluid, with hand action when clicked should unscrew the bottle, pick the pipette set at 1000ul, pipette in cover fluid and pipette out on the strips and to the other wells. Add cover fluid to the well containing the strips and to the other wells. Maintain the level of cover fluid must not over flow. Audio Narration (if any) Description of the action/ interactivity

10. Step 6: T6: Position Electrode assembly 5 2 1 4 3 The user should click on the hand and the events must happen. Show the hand picking the electrodes, placing the electrodes on either side of the tray above the wicks. Redraw the figures Place the electrodes on either side of the tray such that the tip of the electrode touches the paper wick and is immersed in the cover fluid if not add some more oil and close the lid. Audio Narration (if any) ‏ Description of the action/ interactivity

11. Step 7: T7: Switch ON the instrument 5 2 1 4 3 Show the instrument connected with monitor with desktop display. The user should click on icon “IPGphor” the window should appear as shown above. Connect the instrument properly. ON the system and Start the software by clicking on the icon. Audio Narration (if any) ‏ Description of the action/ interactivity

12. Step 8: 5 2 1 4 3 The hand movement with mouse should control the pointer. Pointer click on the protocol tool and a dropdown menu containing “protocol1,protocol 2,protocol 3 must appear and the user should click on the one. Select the specific protocol for the sample to run the IEF. The protocol must be specified and initial run must be performed before going for the final DIGE experiment. T8:Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity

13. Step 9: 5 2 1 4 3 Using the Pointer the user can change the set time, voltage on the protocol. The animator should design in such a way the user can add and change values in the table right side. T8:Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity User can change the protocol according to sample requirements.

14. Step 10: 5 2 1 4 3 The animator must design a dropdown menu for strip length 7cm,18cm 24cm and pI range from 4-7 and 3-10, no of strips from 1-12. Design a Pointer so that the user clicks on the each of tool to set. Redraw the above figure for best feel of the software for the user. T8:Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity User can change the select the strip length and define the number of strips running in the experiment.

15. Step 11: Audio narration Description of the action/ interactivity 5 2 1 4 3 Allow the user to Pointer click on the communication tool to communicate with the instrument and transfer protocol option from software to the system. “a action showing transfer of protocols to instrument should appear on the screen while action going” Then the user should click on the run icon marked with arrow to start the instrument. Once the communication is made and protocol is transferred to instrument. The experiment is ready to focusing. T8:Set program parameters and run IEF

16. Step 12: 5 2 1 4 3 T8:Set program parameters and run IEF Audio narration Description of the action/interactivity The animator should show the graph plotted with Voltage and time according to set protocol (red Line) and a blue line over the red line to track the status of the running protocol denoting that there is no problem during the run. The set protocol is reached, when the sample preparation is perfect and the IEF run will be achieved without any interference.

17. Step 13: Audio narration Description of the action/ interactivity 5 2 1 4 3 In other case: the animator must draw a blue line away from the red line denoting that there is some problem during the run. The set protocol will not reach if the sample preparation contain impurities which may interfere during the run. T8:Set program parameters and run IEF

18. Step 14: Audio narration Description of the action/ interactivity 5 2 1 4 3 If the run is improper the animator should show pointer pausing the run by clicking on the pause button specified in arrow. If the set voltage is not reached, press pause icon to stop the focusing. T8:Set program parameters and run IEF

19. Audio narration Description of the action/ interactivity 5 2 1 4 3 The user should open the lid,remove the electrode and the wicks(the animator should make the white wicks to look yellowish due to impurities. Later animate to replace it with new wicks and then placing back the electrode and closing the lid. In case of improper run replace the wicks with the new one. Step 15: T8:Set program parameters and run IEF

20. Step 16: Audio narration Description of the action/ interactivity 5 2 1 4 3 The animator should point on the run option shown with arrow Restart the run T8:Set program parameters and run IEF

21. Step 17: Audio narration Description of the action/ interactivity 5 2 1 4 3 The animator must continue the drawing blue line from the previous disturbed line over the red line denoting that there is no problem during the run. T8:Set program parameters and run IEF The run continues once the wicks are changed.

22. Step 18: Audio narration Description of the action/ interactivity T9:Mechanism of separation 5 2 1 4 3 The animator should draw the bands with different color lines for proteins close to each other and then show the bands slowly moving down the strip as the protein separates. Bands should stop at one place when the sum of charges is zero. Animation must be for band movement and net charge loss/gain. In an applied electric field proteins stop moving at a point where the net charge on them is zero, at this particular point where point has no-charge is called as Isoelectric point

23. Step 19: Audio narration Description of the action/ interactivity T9:Mechanism of separation 5 2 1 4 3 Animation must be for band movement and net charge loss. For net positive charge the band must loose + ions and for net negative charge the band must loose - ion to become zero net charge after separation. At iso-electric point the number of positive charge is equal to the number of negative charge present on the protein.

24. Step 20: Audio narration Description of the action/ interactivity 5 2 1 4 3 Animate blue line reaching along the red line to show the run is complete. animator should show the pointer clicking on the stop button. Stop the run once the set voltage is reached and the run is completed T10:Completion of IEF

25. Step 21: Audio narration Description of the action/ interactivity T11:Storing the strip 5 2 1 4 3 The animator should show the user action for opening the lid of the instrument by a press on it and the removing the electrodes with user click, and take out the wicks using the forceps. Take out the strips with forceps, drain out the excess cover slip oil on tissue paper by holding the strip with forceps and keeping the vertically. Remove the IPG strip using forceps with proper care must be taken not to bend the strip and excess oil must removed b soaking on the tissue.

26. Step 22 Audio narration Description of the action/ interactivity T11:Storing the strip 5 2 1 4 3 Let user place the strip in the equilibration tray and place it in -20 freezer. The animator should draw a -20’C freezer and the strip holder. Store the strip at -80C until further processing. Once the equilibration buffers are prepared the strip can be taken out to carry out 2D run. Do follow the future viewing IDD for more information.

27. Animation are INTERACTION 1: In Slide-7: let user place the “-” end of strip on “+” side of the instrument and proceeds the setup. Instruction: Display on monitor a very low voltage attained with comparison to set voltage and instruct user to check the strip ends and place it accordingly. NTERACTION 2: In Slide-10: if user place the electrode away from the strip and not exactly on the wicks and proceeds with the setup. Instruction: Display on monitor a very low voltage attained with comparison to set voltage and instruct user to check the setup again to place the electrode properly on the wick and the wick making contact with the gel end. Instructions/ Working area Credits Name of the section/stage: Slide: 7, 8 Interactivity area Tab 01Tab 02Tab 03Tab 04Tab 05Tab 06 Button 01 Button 02 Button 03 Introduction Slide 5 Slide 9 Slide 8 Slide 7 Slide 6 Slide 1 -4 Slide 10

28. Animation area I Instructions/ Working area Credits Name of the section/stage: Slide 9, 10 Interactivity area Tab 08Tab 09Tab 10Tab 11 Button 01 Button 02 Button 03 Tab 07 Slide 24 Slide 22-23 Slide 12-21 Slide 11 Slide 25-26

29. Questionnaire: Question 1 What is use of IEF instrument? a)Holding the strips b) Provide voltage for protein separation c) Prevent protein from denaturation d) Separate protein based on molecular weight Answer: Provide voltage for protein separation Question 2 The component that aid in conductivity and removal of excess salt a)Urea b)Cover fluid c)Wet wicks d)Electrodes Answer: Wet wicks

30. Questionnaire: Question 3 Manifold is made of a) Ceramic b) Plastic c) Gold d) Steel Answer: Ceramic Question 4 What is the Isoelectic point? a)Net charge on the protein is negative b)Net charge on the protein is positive c)Net charge on the protein is zero d)No charge on the protein Answer:Net charge on the protein is zero

31. Questionnaire: APPENDIX 1 Question 5 Charge on the protein at lower and higher pH range is a)Positive and negative charge respectively b) No charge and Negative charge respectively c)Negative charge and no charge respectively d)Positive charge and no charge respectively Answer: Positive and negative charge respectively

32. Links for further reading Reference websites: 2DE Tutorials by Angelika Görg : http://www.wzw.tum.de/blm/deg/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 GE Handbook 2D-Electrophoresis: principle and methods Research papers: Chen JHChen 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.Chang YWYao CW Eymann CEymann C, Dreisbach A, Albrecht D. A comprehensive proteome map of growing Bacillus subtilis cells.Dreisbach AAlbrecht D Maldonado AMMaldonado 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.Echevarría-Zomeño SJean-Baptiste S APPENDIX 2

33. Summary APPENDIX 3 The experiment mostly focussed on the first dimension sepatration of the proteins based on their isoelectric point. The experiment will be successful only when the sample is free of impurities and the run goes without any interference.