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Methodology for the Equilibration of the strips

Separated proteins in the strip has to undergo equilibration step so that the multi-subunit proteins can be separated and prevention of reunion of the separated proteins, stabilization of the separated protein in the gel. Methodology for the Equilibration of the strips.

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Methodology for the Equilibration of the strips

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  1. Separated proteins in the strip has to undergo equilibration step so that the multi-subunit proteins can be separated and prevention of reunion of the separated proteins, stabilization of the separated protein in the gel Methodology for the Equilibration of the strips • Related LOs: IPG strips > Prior Viewing – IDD-6. Extraction of serum protein, IDD-11. Protein quantification, IDD-14. Isoelectric focusing > Future Viewing – IDD-17. SDS-PAGE, IDD-20. Silver staining, • Course Name: Equilibration • 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 the preparations prior to the second-dimension run. • Operate the steps involved in handling the materials and preparation of buffers. • Infer the steps involved to perform the experiment. • Assess the troubleshooting steps involved in the experiments. 2 3 4 5

  3. Definitions and Keywords 1 • Equilibration buffer-1 consists of urea as denaturing agent, SDS, provide uniform negative charge to the protein, DTT , as the reducing agent of the disulphide bond, and glycerol as the stabilizing agent of polyacrylamide gel in the strips. • Equilibration buffer-II consists of urea as denaturing agent, SDS, provide uniform negative charge to the protein, IAA , as the acetylating agent of reduced disulphide bond, glycerol , as the stabilizing agent of polyacrylamide gel in the strips. • Tank buffer consists of Tris base ,glycine, SDS, water which aid proper conductance of current from one terminal to other that helps proper separation of protein. 2 3 4 5

  4. Master Layout 1 Preparation of Equilibration buffer-I (Slide:5 & 7) Preparation of Equilibration buffer-II (Slide:6 & 8) 2 Preparation of Tank Buffer (Slide:9-10) Placing the IPG strip in the equilibration tray (Slide:11) 3 Adding equilibration buffer-I to the strip (12&14) Mechanism (Slide: 13 & 16) Adding equilibration buffer-II to the strip (Slide:15&17) 4 Washing the strips with tank buffer (Slide:18) 5

  5. Audio Narration (if any)‏ Description of the action/ interactivity Step 1: T1:preparation of Equilibration buffer-I 1 2 Weigh urea, 85% glycerol, pH 8.8 1.5M Tris-HCl ,sodium dodecylsulphate, 1% bromophenol blue as per the required volume. 3 Show the bottles labeled as urea, 85% glycerol, pH 8.8 1.5M Tris-HCl, sodium dodecyl sulphate, 1% bromophenol blue( blue solution). Instruct user to place a paper on the measuring balance (display 0.003gm) instruct the user to click ‘tare’ and display shows “0” reading start weighing the reagents one by one like 7.21g urea, 400mg SDS with its name display. Animate, taking out bottle, unscrew the lid, take spatula in one hand, and weigh required amount (to be displayed on RHS). add transfer reagent into empty bottle labeled as Equilibration buffer. In case if the gram exceeds he should remove some quantity or if it is low add to get required amount. Repeat the steps for all other reagent and pipette action like the user should click on the pipette to set to 1000ul and glycerol should be taken added 6 times for 6ml and set 40ul for bromophenol blue and add to the solution Now animate like the user taking the water and measuring 5ml in the cylinder and pouring it in the solution and show like mixing it the solution must look blue. Now ask the user to measure the volume by pouring to the measuring cylinder and the volume has to be made to 20ml.(refer Tris preparation from IDD second dimension slide 11 and 13. Animate like the user taking another tube and transfer 10ml of solution to the new one. 4 5

  6. Description of the action/ interactivity Audio Narration (if any)‏ Step 2: T1:preparation of Equilibration buffer-II 1 2 Weigh 10mg/ml of dithiothreitol and 25mg/ml of iodaoacetamide as per the required volume. 3 Show the bottles labeled as dithiothreitol, Iodoacetamide. Instruct user to place a paper on the measuring balance (display 0.003gm) instruct ‘tare’ and animate a click option on tare button. once display shows “0” reading start weighing 100mg of dithiothreitol and 250mg of iodoacetamide and display in measuring balance. Animate, taking out bottle, unscrew the lid, take spatula in one hand, and weigh required amount (display). Transfer dithiothreitol reagent into tube labeled as Equilibration buffer-I and iodoacetamide into the tube labeled as equilibration buffer II .. 4 5

  7. Step 3 : T2:preparation of Equilibration buffer-I Description of the action/ interactivity Audio Narration (if any)‏ 1 Urea 1.5 M Tris-Hcl Sodium Dodecyl Sulphate 2 85% glycerol 1% Bromophenol blue 3 10 mg/ml Dithiothreitol (DTT) Water Equilibration buffer-I The user should click on the tube to get the constituents of the equilibration buffer –I displayed with audio explanation. Please redraw the figure Equilibration buffer-1 consists of urea acts as denaturing agent, SDS provide uniform negative charge to the protein, DTT acts as the reducing agent of disulphide bond, glycerol as the stabilizing agent of polyacrylamide gel in the strip.strips 4 5

  8. Step 4 : T2:preparation of Equilibration buffer-II Audio Narration (if any)‏ Description of the action/ interactivity 1 Urea 1.5 M Tris-Hcl Sodium Dodecyl Sulphate 2 85% glycerol 1% Bromophenol blue 3 25mg/ml Iodoacetamide (IAA) (DTT) Water Equilibration buffer-I The user should click on the tube to get the constituents of the equilibration buffer –II displayed with audio explanation. Please redraw the figure Equilibration buffer-II consists of urea as denaturing agent, SDS, provide uniform negative charge to the protein, IAA , as the acetylating agent of reduced disulphide bond, glycerol , as the stabilizing agent of polyacrylamide gel in the strips. the strips 4 5

  9. Step 5: T2:Preparation of SDS-buffer 1 Tris base SDS Glycine 2 Water Description of the action Audio Narration 3 Show the bottles labeled as TRIS-Base, Glycine and SDS. The user should click on the required reagent bottle and spatula for weighing. Instruct user to weigh Tris base, let user pick the bottle, uncap it, with help of spatula weigh the 30.3g on a paper over the balance. if the gram exceeds he should remove some quantity or if it low add to get required gram. Follow the same instruction for weighing 144.1 g of glycine and 10g SDS and transfer all the content into beaker. Show the measuring cylinder of 1000ml, animate like the user pouring 500ml of water to the cylinder and then to the beaker containing weighed TrisBase, Glycine and SDS and mix it as shown in slide 15 and instruct the user to pour it in the measuring cylinder and the reading has to be 800ml. The user should click on water to pour 200ml of it in the measuring cylinder For SDS buffer weigh 30.3g of tris base, 144.1g of glycine and 10g SDS accurately. The beaker containing the powder need to be dissolved into the water. 4 Video file: Balancing 5

  10. Step 6: T2: Preparation of SDS-buffer 1 2 Beaker Magnetic bead 3 Description of the action Audio Narration (if any)‏ Prepare 10X SDS running buffer. As an when the powder starts dissolving make up the volume of solution in measuring cylinder to 1000ml by adding water. Show magnetic stirrer instrument. Let user place the beaker on it. Display the beaker containing powder at bottom, liquid layer on top and a magnetic bead at the bottom. Instruct user to ON the instrument, let user cotrol the speed nob and regulate it accordingly to control the mixing speed in the beaker. Animate powder getting into the solution. Show a turbid solution turning colorless Final volume of the solution must be made to 1000ml in measuring cylinder by adding distilled water. 4 5

  11. Step 7 : T3:Placing the IPG strip in the equilibration tray Audio Narration (if any)‏ Description of the action/ interactivity 1 2 3 Place the IPG strip in the equilibration tray with gel side up. Equilibration buffer-I Animate like the user taking the strip from the -20C in a tray by opening the freezer taking out with help of forceps from tray and placing it in equilibration tray as in figure. For information please follow the prior IDD. Please redraw the figures 4 5

  12. Step 8 : T4:Adding equilibration buffer-I to the strip Audio Narration (if any)‏ Description of the action/ interactivity 1 tray 2 3 Add equilibration buffer-1 to the strip and keep on shaker for 15min. The strip must be covered with the buffer. Equilibration buffer-I Zoom equilibration buffer-I tube, and animate like the user setting the pipette to 1000ul and pipetting out the buffer when the user clicks on the pipette (perform two times to get 2000ul)and show the pipette action along the length of the strip as in figure and animate like the user taking the tray and keeping on the rocker and switching on. Display a clock animation for 15min along with movement of tray over the shaker. 4 5

  13. Step 9 : T4:Mechanism of reducing reaction by DTT Description of the action/ interactivity Audio Narration (if any)‏ 1 2 Protein Show reduced form of bond –S-S( put H after each S) 3 Dithiothreitol in equilibration buffer reduces the disulphide bond and helps to maintain all proteins in their fully reduced state. Equilibration buffer-I Show the reaction between the dithiothreitol and disulphide bond of protein. Animate bond breakage. Please redraw the figure 4 5

  14. Step 10 : T4:Remove equilibration buffer-I from the lane. Audio Narration (if any)‏ Description of the action/ interactivity 1 2 3 Remove the strips from the well and place in the new well Equilibration buffer-I After 15min. Animate like the user switching off the shaker and to stop, and show like taking the tray in both hand and holding tray by picking from shaker and using the forceps remove the strips and put in new well. Please redraw the figures 4 5

  15. Step 11 : T5:Adding equilibration buffer-II to the strip Audio Narration (if any)‏ Description of the action/ interactivity 1 2 3 Equilibration buffer-I Zoom equilibration buffer-II tube, and animate like the user setting the pipette to 1000ul and pipetting out the buffer when the user clicks on the pipette (perform two times to get 2000ul)and show the pipette action along the length of the strip as in figure and animate like the user taking the tray and keeping on the rocker and switching on. Display a clock animation for 15min along with movement of tray over the shaker. Add equilibration buffer-2 to the strip and keep in shaker for 15min. The strip must be covered with the buffer. the strips 4 5

  16. Step12 : T5:Mechanism of acetylation reaction by IAA Audio Narration (if any)‏ Description of the action/ interactivity 1 Protein 2 +2 HI 3 Equilibration buffer-I Animate bond breakage reaction followed by acetylation reaction. Animate free acetamide group from IAA going and binding to reduced disulphide bond. Please redraw the mechanism IAA acetylates the reduced disulphide bond and prevent its oxidation, back folding and aggregating of protein subunits. 4 5

  17. Step13 : T5:Remove equilibration buffer-II from the lane. Description of the action/ interactivity Audio Narration (if any)‏ 1 2 3 Remove the strips from the well and place in the new well Equilibration buffer-I After 15min. Animate like the user switching off the shaker and to stop, and show like taking the tray in both hand and holding tray by picking from shaker and using the forceps remove the strips and put in new well. Please redraw the figures 4 5

  18. Step14 : T6:Washing the strips with tank buffer. Audio Narration (if any)‏ Description of the action/ interactivity 1 2 3 Wash the strips with tank buffer after second equilibration step. Now the strip is ready for SDS-PAGE run. For more information and continuity please go through future IDD. Equilibration buffer-I Take out the strip from the tray with forceps in one hand. The user should set the pipette to 1000ul and Take out 1000ul of SDS buffer with help of pipette. Now pipette out tank buffer along the strip. Animate the process. Please redraw the figures 4 5

  19. Button 01 Button 02 Button 03 Slide 12&14 Slide 9-10 Slide 11 Slide 13&16 Slide 5 &7 Slide 6 & 8 Introduction Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Animation area In slide-12: what if user forgets to add Equilibration buffer-I and proceeds further. Instruction: animator must connect the user to scanned gel image showing lot of horizontal blue lines on the gel. In slide-18: after equilibration step and 2D run, show a gel image with lines (streaks), spots only on ends not in the middle, ask for user input. Instruction: provide choice like: A) uneven equilibration (spots only on ends not in the middle). B) less concentration of DTT/IAA (horizontal lines). user must be able to find the right choice. Interactivity area Instructions/ Working area Credits

  20. Button 01 Button 02 Button 03 Slide 18 Slide 15&17 Tab 07 Tab 08 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Animation area Interactivity area Instructions/ Working area Credits

  21. Questionnaire: APPENDIX 1 Question 1 The action of DTT in equilibration buffer 1 is • Oxidation • Reduction • Sulfation • Acetylation Answer: Reduction Question 2 The action of IAA in equilibration buffer 2 is • Oxidation • Reduction • Sulfation • Acetylation Answer:Acetylation

  22. Questionnaire: APPENDIX 1 Question 3 The reagent glycerol is added for • Washing the strips • Stabilizing the gel in the strip • Separation of the gel from the strip • Smoothness of the stri[ Answer:Stabilizing the gel in the strip Question 4 DTT and IAA acts on • Electovalent bond • Covalent bond • Disulphide bond • Hydrogen bond Answer: Disulphide bond

  23. Questionnaire: APPENDIX 1 Question 5 Tank buffer consists of • Tris base • Glycine • SDS • All the above Answer: all the above

  24. Summary: APPENDIX 1 Equilibration of the strips is the important step that aid in proper separation of the proteins in second dimension SDS-PAGE. Improper equilibration will result in streaking and protein precipitating in the gel forming a vertical streaking Equilibration solutions has to be fresh to ensure the proper ingredients are present which aid in producing good quality gels

  25. APPENDIX 2 Links for further reading • Reference websites: 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 GE Handbook 2D-Electrophoresis: principle and methods Research papers: Chen JH, Chang YW, Yao CW et al. Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.ProcNatlAcadSci U S A2004, 7;101(49):17039-44. Eymann C, Dreisbach A, Albrecht D. A comprehensive proteome map of growing Bacillus subtilis cells. 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.

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