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Methodology for Silver Staining

Methodology for Silver Staining.

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Methodology for Silver Staining

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  1. Methodology for Silver Staining Proteins that are separated by the 2-DE has to be stained by the staining solution for visualization. Many staining methods are available for visualization of proteome, choosing appropriate for the detection of low concentration protein is essential for the analysis` ‏ • Related LOs: Staining techniques. > Prior Viewing – IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE > Future Viewing – IDD-22. 2D-gel scanning and image analysis, IDD-26. Spot picking • Course Name: Methodology for silver staining • 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 preparation steps for staining • Identify the mechanism behind the staining • Operate the steps involved in getting good proteome profile • Infer the steps involved to perform the experiment • Assess the troubleshooting steps involved in the experiments 2 3 4 5

  3. Fixation (Slide: 5-6) Sensitization (Slide: 7-10) Staining (Slide: 11-15) Developing (Slide: 16-18) Stopping (Slide: 19-20) Preservation (Slide: 21-23) Master Layout 1 2 3 4 5 http://www-lmmb.ncifcrf.gov/plasmaDB/plasmaLlbl.html Animate for user click to show images or instruments used for each step from the respective slides.

  4. Definitions and Keywords 1 1) Fixing solution: consists of ethanol, glacial acetic acid and water. The solution precipitates the protein in the gel. 2) Sensitizing solution: consists of ethanol , Sodium thiosulphate water, glutaraldehyde. 3) Staining solution: containing silver bind to the protein and developing solution containing formaldehyde and sodium carbonate where the silver is reduced to metallic silver. 2 3 4 5

  5. Step 1: T1: Fixation 1 FIXING SOLUTION 2 WATER 3 Description of the action/ interactivity Audio Narration (if any)‏ Show the bottles labeled as ethanol, glacial acetic acid, measuring cylinder (MC) and water. Instruct user to click on bottle and allow him to pick each bottle to pour 75ml of ethanol in 100ml MC, 25 ml glacial acetic acid in 50ml MC, each at a time to 250ml MC. now instruct the user to pour water till the level of water reaches 250ml in the 250ml measuring cylinder. In case if the level is more, instruct user to remove the extra solution accordingly. Animate like pouring and increase in the level of solution simultaneously in the measuring cylinder. Later let user transfer the solution into the fixing solution bottle. Please re-draw the figure. Prepare fixing solution by measuring 75ml of ethanol, 25 ml glacial acetic acid and make up the volume to 250ml with water. 4 5

  6. Description of the action/ interactivity Audio Narration (if any)‏ Instruct user to pour fixing solution into the staining tray. when the user clicks on the hand and allow user to pick the gel from the 2D run, take out the gel from the glass plate and transfer it into the tray or keep the galssplate in the tray and transfer the gel gently. Now animate like the user taking the tray containing gel when the user clicks on the hand and place it on ROCKER. Allow user to switch on the rocker and animate like moving a knob on the instrument adjustment for speed, when the user clicks on the hand to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray. Step 1: T1: Fixation 1 2 GEL Staining tray Rocker 3 Place the gel in the fixing solution to remove the compounds like SDS, ampholytes that produces background for 120 minutes with gentle shaking. In the meantime prepare the reagent for the next step. 4 5

  7. Description of the action/ interactivity Audio Narration (if any)‏ Let user prepare sensitizing solution (SS). Instruct user to place a paper on the measuring balance (display 0.003gm) and animate a click option on tare button when the user clicks on the hand. Show the display showing “0” reading. Animate, taking the bottle labeled as sodium acetate, unscrew the lid, take spatula in one hand, and weigh 17g amount, transfer to SS bottle. In case if the gram exceeds he should remove some quantity or if it is low add to get required amount. Instruct the user to pour water till the level of water reaches 250ml in the measuring cylinder. Later transfer the solution into the sensitizing solution bottle and show like mixing as shown in slide 9 Step 2: T2: sensitizing 1 SENSITIZING SOLUTION 2 Sodium acetate 3 Prepare sodium acetate solution required to prepare sensitizing solution (SS). 4 5 Video file: Balancing

  8. Step 2: T2: sensitizing 1 2 Beaker Magnetic bead 3 Audio Narration (if any)‏ Description of the action The reagent solution need to be properly mixed with help of magnetic stirrer instrument. 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 control the speed knob and regulate it accordingly to control the mixing speed in the beaker. Animate powder getting into the solution. Show a turbid solution turning colorless 4 Video file: Magnetic stirrer 5

  9. Description of the action/ interactivity Audio Narration (if any)‏ Show the bottles labeled as ethanol, 5% sodium thiosulphate, 25% glutaraldehyde, measuring cylinder (MC) and water. Instruct user to click on bottle and allow him to pick each bottle to pour 75ml of ethanol in 100ml MC, add 30ml of sodium acetate solution in 50ml MC, 10 ml of 5% w/v Sodium thiosulphate amount in 50ml MC and transfer each into 250ml MC. In case if the level is more, instruct user to remove the extra solution accordingly. Animate pouring and increase in the level of solution simultaneously in the measuring cylinder and instruct the user to pour water till the level of water reaches 250ml in the measuring cylinder. animate like the user taking the pipette and setting to 1000ul and measuring the 1000ul of glutaraldehyde and pouring it in the sensitizing solution bottle and again set the pipette to 250ul and follow the same step. Transfer the solution from 250ml MC to SS bottle Step 2: T2: sensitizing 1 SENSTIZINGSOLUTION 2 WATER 3 Prepare sensitizing solution by measuring 75ml of ethanol,30ml sodium acetate, 10 ml of 5% w/v sodium thiosulphate and make up the volume to 250ml with water. Mix properly to dissolve the sodium thiosulphate completely. later add the 1.25 ml of 25% w/v glutaraldehyde before use. 4 5

  10. Step 2: T2: sensitizing 1 SENSTIZINGSOLUTION 2 WASTE Rocker 3 Description of the action/ interactivity Audio Narration (if any)‏ Instruct user to stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. Now pour sensitizing solution into the staining tray and allow user to pick the gel and transfer it into the tray. Now animate like the user taking the tray containing gel when the user clicks on the hand and place it on ROCKER. Allow user to switch on the rocker and animate like moving a knob on the instrument adjustment for speed. Animate see-saw movement for the rocker along with solution movement in the tray Place the gel in the sensitizing solution for 120 minutes with gentle shaking. In the meantime prepare the reagent for the next step. 4 5

  11. Step 3: T3:staining 1 SILVERSOLUTION 2 WATER 3 Description of the action/ interactivity Audio Narration (if any)‏ Show the bottles labeled as 2.5% Silver nitrate, measuring cylinder and water. Instruct user to click on silver nitrate bottle and allow him to pick the bottle to pour in 25ml in 50ml MC and later transfer to 250ml MC to makeup the volume with water. In case if the level is more, instruct user to remove the extra solution accordingly. Animate like pouring and increase in the level of solution simultaneously in the measuring cylinder and instruct the user to pour water till the level of water reaches 250ml in the measuring cylinder . Later transfer the solution into the silver solution bottle. Prepare silver solution by measuring 25ml of 2.5%w/v silver nitrate solution and make up the volume to 250 ml by adding water. 4 5

  12. Step 3: T3:staining 1 DISTILLED WATER 2 WASTE Rocker Description of the action/ interactivity Audio Narration (if any)‏ 3 Instruct user stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. Now pour Distilled water into the staining tray and allow user to pick the gel and transfer it into the tray. Now animate like the user taking the tray containing gel and placed on ROCKER when the user clicks on the hand. Allow user to switch on the rocker and animate like movement on the knob on the instrument for speed adjustment. Animate see-saw movement for the rocker along with solution movement in the tray. Show a clock running for 15 minutes and show like repeating this step for 5 times Place the gel in the water for 15 minutes with gentle shaking and repeat the step for 5times 4 5

  13. Description of the action/ interactivity Audio Narration (if any)‏ Instruct user stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. . Now pour silver solution into the staining tray and allow user to pick the gel and transfer it into the tray. Now animate like the user taking the tray containing gel and placed on ROCKER. Allow user to switch on the rocker when the user clicks on button and animate like moving a knob on the instrument for speed adjustment. Animate see-saw movement for the rocker along with solution movement in the tray. Show the clock running for 120 minutes Step 3: T3:staining 1 SILVERSOLUTION 2 WASTE Rocker 3 Place the gel in the silver solution for 120 minutes with gentle shaking. 4 5

  14. Description of the action/ interactivity Audio Narration (if any)‏ Animate to zoom the some part of gel to show on top of it moving circle in which “Ag” has to be prescribed, these Ag circles must go and bind to protein molecules within the gel. the user should click on the tray to get the reaction. Please redraw the figure Step 3: T3:staining 1 2 3 Place the gel in the staining solution followed by developing solution containing formaldehyde and sodium carbonate where the silver is reduced to metallic silver which starts glowing. 4 5

  15. Step 3: T3:staining 1 DISTILLED WATER 2 WASTE Rocker Description of the action/ interactivity Audio Narration (if any)‏ 3 Instruct user to stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. Now pour Distilled water into the staining tray and allow user to pick the gel and transfer it into the tray. Now animate like the user taking the tray containing gel and placed on ROCKER when the user clicks on the hand . Allow user to switch on the rocker and animate like moving a knob on the instrument for speed adjustment. Animate see-saw movement for the rocker along with solution movement in the tray. Show a clock running for 1 minutes and show like repeating the step for 5 times Place the gel in the water for 1 minutes with gentle shaking and repeat the step for 5times to remove the silver solution 4 5

  16. Description of the action/ interactivity Audio Narration (if any)‏ Step 4: T4: DEVELOPING 1 DEVELOPINGSOLUTION 2 Sodium carbonate 3 Instruct user to place a paper on the measuring balance (display 0.003gm) and animate a click option on tare button when the user clicks on the hand. Show the display showing “0” reading. Animate, taking the bottle labeled as sodium carbonate, unscrew the lid, take spatula in one hand, and weigh 6.25g amount. In case if the gram exceeds he should remove some quantity or if it is low add to get required amount. Now transfer the weighed amount to 250ml MC. Instruct user to pour water till the level of water reaches 250ml in the measuring cylinder. Later transfer the solution into the sensitizing solution bottle and show like mixing as shown in slide 9 Prepare developing solution by measuring 6.25g of sodium carbonate 4 5

  17. Description of the action/ interactivity Audio Narration (if any)‏ Show the bottles labeled as 37% of formaldehyde, measuring cylinder and water. animate like the user taking the pipette and setting to 200ul and measuring the 200ul of formaldehyde and pouring it in the developing solution bottle. show the mixing as in slide: 9. Step 4: T4:Developing 1 DEVELOPINGSOLUTION 2 WATER 3 To the solution containing sodium carbonate add 37% of 0.2 ml formaldehyde just before use. 4 5

  18. Description of the action/ interactivity Audio Narration (if any)‏ Instruct user stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. Now pour developing solution into the staining tray and allow user to pick the gel and transfer it into the tray. Now animate like the user taking the tray containing gel and place it on ROCKER when the user clicks on the hand. Allow user to switch on the rocker and animate like moving a knob on the instrument adjustment for speed, when the user clicks on the hand to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray. Show a clock running for 5-6 minutes, animate spots appearing on the gel and user taking the try for discarding. Step 4: T4:Developing 1 DEVELOPINGSOLUTION 2 WASTE Rocker 3 Place the gel in the developing solution for 5-6minutes with gentle shaking. Stop the shaking once sufficient spots are visible at required intensity. 4 5

  19. Description of the action/ interactivity Audio Narration (if any)‏ Step 5: T5:Stopping 1 STOPSOLUTION 2 EDTA-Na2 3 Instruct user to place a paper on the measuring balance (display 0.003gm) and animate a click option on tare button when the user clicks on the hand. Show the display showing “0” reading. Animate, taking the bottle labeled as EDTA-Na2 , unscrew the lid, take spatula in one hand, and weigh 3.65g amount. In case if the gram exceeds he should remove some quantity or if it is low add to get required amount. Instruct user to pour water till the level of water reaches 250ml in the 250ml MC. Later transfer the solution into the stop solution bottle and show like mixing as shown in slide:9. Prepare stop solution by measuring 3.65 g of EDTA-Na2 4 5 Video file: Balancing

  20. Step 5: T5:Stopping 1 STOPSOLUTION 2 WASTE Rocker 3 Description of the action/ interactivity Audio Narration (if any)‏ Instruct user stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. Now pour stop solution into the staining tray and allow user to pick the gel and transfer it into the tray. Now animate like the user taking the tray containing gel and placed on ROCKER when the user clicks on the hand . Allow user to switch on the rocker and animate like moving a knob on the instrument adjustment for speed ,when the user clicks on the hand and to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray. Show a clock running for 120 minutes Place the gel in the stopping solution until the spots reach desired intensity. In the meantime prepare the reagent for the next step. 4 5

  21. Description of the action/ interactivity Audio Narration (if any)‏ Show the bottles labeled as 87% glycerol, measuring cylinder and water. Instruct user to click on glycerol bottle and allow him to pick, to measure 25ml in 50ml MC and transfer to 250ml MC. In case if the level is more, instruct user to remove the extra solution accordingly. Animate pouring and increase in the level of solution simultaneously in the measuring cylinder. and instruct the user to pour water till the level of water reaches 250ml in the measuring cylinder Later transfer the solution into the preservation solution bottle. Step 6: T6:Preservation 1 PRESERVATION SOLUTION 2 WATER 3 Prepare preservation solution by measuring 25ml of 87% glycerol and 250 ml of water and mix them well. 4 5

  22. Description of the action/ interactivity Audio Narration (if any)‏ Instruct user stop the rocker, pick the tray, discard the liquid with user action by tilting the tray or take out the cap of the outlet at the bottom of the tray. Now pour preserving solution into the staining tray and allow user to pick the gel and transfer it into the tray.. Now animate like the user taking the tray containing gel and placed on ROCKER when the user clicks on the hand . Allow user to switch on the rocker and animate like moving a knob on the instrument adjustment for speed ,when the user clicks on the hand and to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray. Step 6: T6:Preservation 1 Preservation solution 2 WASTE Rocker 3 The silver stained protein spots can be seen on the gel, which can be preserved using treatment with preservation solution. 4 5

  23. Description of the action/ interactivity Audio Narration (if any)‏ Instruct user to stop the rocker, pick the tray, discard the liquid with user action. Display gel having spots all around the gel. Step 6: T6:Preservation 1 http://www.bio-rad.com/prd/en/US/adirect/biorad?ts=1&cmd=BRCatgProductDetail&vertical=LSR&catID=09c4382c-f801-4c0a-827b-ac972ced3969 2 WASTE 3 Now the stained gel is ready for scanning and analysis. For more information do follow the future viewing IDD mentioned in Slide:1 4 5

  24. Button 01 Button 02 Button 03 Slide 7-10 Slide 19-20 Slide 11-15 Slide 16-18 Slide 21-23 Slide1-4 5-6 Introduction Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Animation area • In Slide-19: animate the stained gel with all dark intensity bands and streaking all over the gel region. let user makes a choice from the options. Instruction: a) developing step kept for more time: add the stop solution as soon as the spots are appeared. b) Stop solution kept for too long c) preservation step kept for too long d) Washing step kept for too long Interactivity area Instructions/ Working area Credits

  25. Questionnaire: APPENDIX 1 Question 1 Important ingredient in this staining technique • Silver • Formaldehyde • Ethanol • Water Answer: Silver Question 2 In this technique silver undergo a)Oxidation b)Reduction c)Condensation with protein d)Evaporation Answer: Reduction

  26. Questionnaire: APPENDIX 1 Question 3 In this technique Formaldehyde undergoes a)Oxidation b)Reduction c)Condensation with protein d)Evaporation Answer: oxidation Question 4 The silver and formaldehyde is converted to a)Metallic silver and formic acid b) Silver oxide and formic acid c)Silver and methanol d)Both a and b Answer:a)Metallic silver and formic acid

  27. Questionnaire: APPENDIX 1 Question 5 The technique detects protein of concentration in the range of a)Micrograms b)Milligrams c)Nanograms d) Kilograms Answer: c)Nanograms

  28. 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 Healthcare: 2D Electrophoresis, Principles and methods by Angelika Görg , 3rd edition. 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.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. 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.

  29. APPENDIX 3 Summary The method is more sensitive, with any variation in ingredients will affect the staining efficiency and has the ability to detect the nano gram level of proteins. The staining process can be modified for mass spec compatibility and the method involves fixiation,sensitization,washing, staining,developing ,stopping ,preserving the gel to get the desired result

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