1. Off Gel Fractionator Proteins exhibit unique isoelectric property for a particular pH, at which the net charge on protein is zero. The isoelectric property of the proteins are exploited in the first dimension separation by off gel fractionator ‏ Related Los: proteins isoelectric property, amphoteric molecules, IPG strips, rehydration buffer > Prior Viewing - IDD-11. Protein quantification, IDD-13. Cyanine dye labeling > Future Viewing – IDD-16. Equilibration of IPG strips, IDD-17. SDS-PAGE  Course Name: Isoelectric Focussing/IEF/1D saparation  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 the experiment. 5. Assess the troubleshooting steps involved in the experiments. 5 3 2 4 1

3. Master Layout 5 3 2 4 1 Place rehydrated IPG strip (Slide:5-9) Sample loading (Slide:10-11) Position Electrode assembly (Slide:12-14) Switch ON the instrument (Slide:15) Set program parameters and run IEF (Slide:16-22) Mechanism of separation (Slide:23-25) Collecting of fractions (Slide:26)

4. Definitions and Keywords 1. Protein: Proteins 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 ions, salts, buffer and aid in conductivity. 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:Place rehydrated IPG strip 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user takes manifold, a tissue paper from the rack and keep it on working table. Let user pick the tissue and place it on the manifold to make a movements for cleaning. Animate manifold image from wet to final dry image once user is done with cleaning. 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 1: T1:Place rehydrated IPG strip 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user takes IPG strips box from -20'C freezer. From the box, a pack of strips need to taken out, let user opens the packing seal, take out the strip with forceps. Let user open the backing cover from the strip, and place the strip which has gel side up in one lane of manifold as shown in the figure. Let + end of strip be placed on left side of manifold. Let user makes a note of strip number, printed on backside of strip. IPG strips help to carry out 1D separation. The strip has a backing cover, which need to be removed before use. And gel must face up while carrying out 1D separation.

7. Step 1: T1:Place rehydrated IPG strip 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user takes out the well frame, and places it on the IPG strip. Let user keeps the right side of the frame little elevated, place the left side of the frame against the mechanical stop (about 2 cm, from the end of the tray). Let user apply little pressure to fix the well frame properly down until it snaps in. The well frame have small compartment, where in sample is loaded, this helps to hold the sample on top of strip. Be careful not to move or crimp the strip while placing the frame down.

8. Step 1: T1:Place rehydrated IPG strip 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Now let user takes out rehydration buffer tube from the freezer, thaw the tube with user interaction, pick 200ul pipette, set it for 40ul and pipette in 40ul of rehydration buffer in each well. Let user tap the tray onto the desk, to ensure that the Rehydration buffer reaches the gel. Animate clock for 15min and change the appearance of colorless strip turning into blue color. IPG strip need to be swell with rehydration buffer for 15min. Once the rehydration buffer has wetted the gel surfaces place the wicks.

9. Step 1: T1:Place rehydrated IPG strip 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity From the kit let user takes out labelled Electrode Pads, remove four pads for each IPG strip. Using forceps, wet one electrode pad by pipetting 0.5ml of rehydration buffer using 1ml pipette set for 0.5ml. Place one wetted electrode pad on each end of the IPG strip. Place a second wetted electrode pad on top of the first electrode pad. User should make sure that there is no gap between the pad and the well frame. The upper pads should be replaced every 24 hours of fractionation.

10. Step 2: T2: Sample loading 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user takes out the sample solution from the freezer. Thaw the tube with user interaction, once the sample turns to liquid form instruct user to load the sample in wells. Let user pick 200ul pipette, set it for 150ul and pipette in 150ul of sample in each well. Each time sample is added check for the leak if any between the wells. If so remove the well frame and apply pressure and fix it properly with user interaction. Sample solution is mixture of sample and Protein OFFGEL Stock Solution. Add 150ul of sample in each well. Check for any leaks outside the wells. Sample must be retained into the wells.

11. Step 2: T2: Sample loading 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user place the cover seal over the well frame and press down gently on each well to secure proper fit. The cover seals have proper groves to get fit into the wells tightly. Seal cover avoids evaporation, be careful not to move the frame in the tray while applying cover seal.

12. Step 3: T3: Position Electrode assembly 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user reapply 10μL of dH 2 0 onto the electrode pads at each of the IPG gel ends. Be careful not to move the pads. To be on safer side to avoid wicks from drying add 10μL of dH20.

13. Step 3: T3: Position Electrode assembly 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Instruct user to pipette cover fluid (mineral oil) onto the gel strip ends. Let user pick 200μl of pipette to pipette in 200μL cover fluid onto the anode end (fixed electrode) of the IPG strip. Similarly pipette 400μL cover fluid at the cathode side (movable Electrode). Let user place the tray on the instrument platform. When pipetting cover fluid, use caution not to move the electrode pads. The cover fluid should not extend higher than 1/2 the height of the tray grooves

14. Step 3: T3: Position Electrode assembly 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Instruct user to place electrode. Let user pick anode electrode (fixed electrode) to place into the slots on the left side of the tray. Now let user pick cathode electrode(movable) and place into the slots on the right side of the tray. The movable electrode should touch the end of the frame. Be sure that the moveable electrode makes contact with the electrode pad. If not, gently reposition the electrode pad Anode electrode Cathode electrode

15. Step 4: T4: Switch ON the instrument 5 2 1 4 3 Audio Narration (if any) ‏ Description of the action/ interactivity Let user close the lid of the instrument and ON the instrument and set the focusing protocol in the software. The focusing protocol is the main part of the experiment.

16. Step 5: T5: Set program parameters and run IEF 5 2 1 4 3 Audio Narration (if any) ‏ Text to be displayed (if any) ‏ (DT) Description of the action/ interactivity 1 Method selection 2 Start/Stop button 3 Tray corona 4 Status indicator 5 Method step 6 Instrument lock 7 Run parameters 8 Actual values 9 Actuals per lane

17. Step 5: 5 2 1 4 3 Redraw the software display like in previous slide. Let the display reacts for the user interaction. Instruct user to edit the status, a new window like above must pop up for user to feed in the values. And let user click on apply to save the setting. The parameter setting are for the focusing step. Depending upon the strip length used and user requirement, the parameters can be changed. T5: Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity

18. Step 5: 5 2 1 4 3 T5: Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity Audio Narration (if any) ‏ Description of the action/ interactivity 1 Method selection 2 Parameter per step 3 Timtable graphic 4 Parameter in Timetable 5 Addition of Timetable lines 6 Save/Delete options

19. Step 5: 5 2 1 4 3 Redraw the software display like in previous slide. Let the display reacts for the user interaction. Instruct user to add parameters for HOLD step. And let user click on apply to save the setting for method selection. And click on run button to start the off gel fractionator. The parameter setting are for the hold step. The timeline displays the set voltage, current and volthour with respect to time. T5: Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity

20. Step 5: 5 2 1 4 3 Display like above soon after the run button is clicked. Once run starts, the animator must plot display the volt attaining as the time passes. T5: Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity The graph indicates the run status. If any problem during the focusing the volt will not attain the set voltage, which will be displayed in the graph plot.

21. Step 5: 5 2 1 4 3 If the set voltage is not achieved. Animator should display it on graph. Instruct user to stop the run. zoom to show wick turned to yellow color. Instruct user to replace it with fresh one. Once user replace it with fresh one, instruct him to click on run button. T5: Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity In case if there is problem in run, replace the top wick with fresh one and start the run again.

22. Step 5: 5 2 1 4 3 If the set voltage is not achieved. Animator should display it on graph. Instruct user to stop the run. Display error “electrode not connected”. Let user fix the electrode again and fit it properly. Once done instruct user to click on RUN button. T5: Set program parameters and run IEF Audio Narration (if any) ‏ Description of the action/ interactivity In case if there is problem in run, check for the electrode position. Reconnect the electrode properly and run again.

23. Step 6: Audio narration Description of the action/ interactivity T6:Mechanism of separation 5 2 1 4 3 The animator should draw the bands with different color lines for proteins close to each other. After applying the voltage 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 done for band movement with net charge loss/gain. Protein stops moving at a point when the net charge is zero which is called as Isoelectric point

24. Step 6: Audio narration Description of the action/ interactivity T6: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. Protein stops moving at a point when the net charge is zero which is called as Isoelectric point

25. Step 6: 5 2 1 4 3 In case of OFF gel Fractionator, animate the above steps. Red and blue colored circles are two different types of proteins. During high voltage animate the circles must start moving into the well through the gel as shown above. Once the focusing is done all red circles must be in one well and blue ones in other well. T6:Mechanism of separation Audio Narration (if any) ‏ Description of the action/ interactivity In case if there is problem in run, replace the top wick with fresh one and start the run again.

26. Step 7: 5 2 1 4 3 Once the run is over, instruct user to stop the run, and let user open the lid, seal cover and pick the pipette to take out the sample from each well and transfer to fresh tube and label accordingly. T7:Collecting of fractions Audio Narration (if any) ‏ Description of the action/ interactivity The fraction collected after the run can be taken for further analysis for more information please go through through future viewing IDD.

27. Animation area INTERACTION 1: In Slide-9: if user place the electrode pad on the strip backing support rather then on the gel and proceeds 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 pad properly on the gel. INTERACTION 2: In Slide-13: while applying the cover fluid, in a hurry or to test the equipment, let user pour the fluid to a lesser quantity 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 pour sufficient fluid to cover the strip and the fluid level making contact with electrode. Instructions/ Working area Credits Name of the section/stage Interactivity area Tab 01Tab 02Tab 03Tab 04 Tab 05Tab 06 Button 01 Button 02 Button 03 Introduction Slide 5- 9 Slide 16-22 Slide 15 Slide 12-14 Slide 10-11 Slide 1-4 Slide 23-25 Tab 07 Slide 26

28. Questionnaire: APPENDIX 1 Question 1 For a 24cm strip 24 well, what is the total protein load (offgel stock solution+sample)? a) 2400ul b) 3600ul c) 2424ul d) 3000ul Answer: b) 3600ul Question 2 For a 12cm strip 12 well, what is the total protein load (offgel stock solution+sample)? a) 2000ul b) 1800ul c) 1212ul d) 3000ul Answer: b) 1800ul

29. Questionnaire: APPENDIX 1 Question 3 The Glycerol is used for 1) Reduces electroendosmotic effects 2) Prevents drying of gel 3) Prevents urea crystallization a)I Only b)II Only c)III only d)I, II and III Answer: d) I, II and III Question 4 Bromophenol blue allows? a) Control of liquid distribution b) To color the protein c) To remove the protein charge d) To add charge on the protein Answer: a) Control of liquid distribution

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

31. Links for further reading Reference websites: GE Handbook 2D-Electrophoresis: principle and methods Research papers: 1.http://www.chem.agilent.com/Library/brochure s/5990-5596EN.pdf 2.https://intramural.nhlbi.nih.gov/Cores/PAF/Doc uments/Instruction%20for%20OFFGel.pdf 3. Jerome Chenau et, al., Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling. Proteome Science 2008, 6:9 APPENDIX 2

32. Summary APPENDIX 3 The experiment mostly focuses on the first dimension separation of the proteins based on their isoelectric point. The process differs from conventional gel electrophoresis, as in that the sample components separated as fractions can be recovered after electrophoresis. The experiment will be successful only when the sample is free of impurities and the run goes without any interference.