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Phospho Staining

Phospho Staining. The experiment mainly focuses on the staining of proteins which are phosphorylated during the post translational modification. Related LOs: Staining techniques.

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Phospho Staining

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  1. Phospho Staining The experiment mainly focuses on the staining of proteins which are phosphorylated during the post translational modification • Related LOs: Staining techniques. > Prior Viewing – IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE, IDD-18. Second dimension separation of proteins, IDD:33 Western blot assay. > Future Viewing – IDD-22. 2D-gel scanning and image Analysis, IDD-26. Spot picking • Course Name: Methodology for Phospho staining • 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 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:5-7) Sensitization (Slide: 8-10) Staining (Slide: 11-13) Developing (Slide:14-19) Stopping (Slide: 20-25) Preservation (Slide: 26-27) Master Layout 1 2 3 4 5

  4. Definitions and Keywords 1 • Pro-Q diamond stain : fluorescent dye that stains the phosphorous group in the protein that can be easily seen when the gel/membrane exposed to the UV light/LASER. • Fixing solution: consists of ethanol, glacial acetic acid and water. The solution helps to precipitate the protein in the gel. 2 3 4 5

  5. Description of the action Audio Narration (if any)‏ Step 1: T1: Fixation 1 2 Once proteins are separated on SDS-PAGE, they need to be transferred onto PVDF membrane by Electroblotting process. After electro-blotting of proteins on to the membrane, which later need to be dried to carry out staining process. Animator explain the process with the separation of sample by SDS-PAGE, transfer of gel proteins onto a PVDF or nitrocellulose membrane by standard procedures like explained in IDD:33 Western blot assay. Instruct the user to go through prior viewing IDDs or display the images from the same at a glance. 3 4 5

  6. Description of the action Audio Narration (if any)‏ Step 1: T1: fixation 1 2 3 Prepare the fixing solution and transfer it to the tray. Fixation step helps to fix the proteins, remove the excess SDS which may interfere in the staining process and also modify the proteins to enhance the staining reaction. Animate bottles labeled as Fixing solution, concentrated acetic acid, deionized water (dH2O), methanol, measuring cylinder MC, let user picks up these from the rack. Instruct user to prepare Fixing solution. Add 7mL of concentrated acetic acid in 10ml MC to 80mL deionized water (dH2O) in 100ml MC, add 10mL of methanol, bring the volume up to 100mL with dH2O, and mix thoroughly. Animate the steps with user control. 4 5

  7. Step 2: T1: fixation 1 2 Description of the action Audio Narration (if any)‏ 3 Animate like the user takes the PVDF membrane after electroblotting. Animate like user takes the membrane and put it in the tray containing fixing solution and show a clock running for 10 minutes Place the membrane in the fixing solution for 10 minutes on a shaker. 4 5

  8. Description of the action Audio Narration Step 8: T2: Sensitization 1 2 gel 3 Animator should instruct the user to discard the previous liquid into the waste by tilting the tray or taking out the cap from the opening at the bottom of the tray. now place the gel in the ultra-pure water and allow it incubate 10 minutes Show a clock running 10 minutes Place the gel in ultrapure water for 10 minutes. Washing is carried out to remove the fixing solution. Washing can be done two or three times, each time with fresh water. 4 5

  9. Description of the action Audio Narration (if any)‏ Step 9: T2: Sensitization 1 2 3 Instruct user to prepare Reagent:1. Place the required materials on the table, like Sulfosalicylic acid bottle, acid, water and measuring cylinder. Let user measure 10ml of Sulfosalicylic acid, transfer it to beaker, now measure 90ml of water in 100ml MC and add to beaker. Let user pick the beaker and shake it to mix the solution. Reagent 1 helps to remove unwanted free and low molecular weight phospho compounds if any from the PVDF membrane. 4 5

  10. Description of the action Audio Narration Step 9: T2: Sensitization 1 2 3 Transfer the gel to the tray containing sulfosalicylic acid and allow it incubate for 15 minutes. Animator should draw a bottle labeled as “reagent 1 Sulfo salicylic acid. Animator should instruct the user to discard the previous liquid into the waste by tilting the tray or taking out the cap from the opening at the bottom of the tray. Instruct the user to pour the reagent:1 to the tray containing the gel and leave it for 15 minutes Show a clock running 15 minutes 4 5

  11. Description of the action Audio Narration (if any)‏ Step 10: T3: Staining 1 2 3 Animate bottles labelled as Reagent 2, Sulfosalicylic acid, deionized water (dH2O); Cacl2; measuring cylinder 100 mL. Instruct user to prepare Reagent 2. Add 10ml of Sulfosalicylic acid acid to 80 mL deionized water (dH2O) in 100ml MC; add 10 mL of Cacl2; bring the volume up to 100 mL with dH2O; and mix thoroughly. Animate the steps with user control. Reagent 2 helps to increase band sharpness. 4 5

  12. Description of the action Audio Narration Step 10: T3: Staining 1 2 3 Transfer the gel to the tray containing sulfosalicylic acid and calcium chloride and allow it incubate for 15 minutes Animator should draw a bottle labeled as “reagent 2 Sulfo salicylic acid+ Calcium Chloride. Animator should instruct the user to discard the previous liquid into the waste by tilting the tray or taking out the cap from the opening at the bottom of the tray. Instruct the user to pour the reagent:2 to the tray containing the gel and leave it for 15 minutes. Show a clock running 15 minutes 4 5

  13. Description of the action Audio Narration Step 11: T3: Staining 1 2 3 Animator should draw a bottle labeled as “ultrapure water” let user discard the previous liquid into the waste. Let user pick ultrapure water, measure around 250ml and pour into the tray containing the gel and leave it for 5min. Transfer the gel to the tray containing ultrapure water for washing. Washing is carried out to remove residual solution from previous step. 4 5

  14. Description of the action Audio Narration (if any)‏ Step 12: T4: Developing 1 2 3 Instruct user to prepare Reagent:3. Place the required materials on the table, like weighing balance, NaOH bottle, water and measuring cylinder. Let user weigh 20g of NaOH, transfer it to beaker, now measure 1L of water and add to beaker. Let user pick the beaker and shake it to mix the solution. Reagent:3 helps in hydrolysis to free more phosphate for staining. 4 5

  15. Description of the action Audio Narration Step 12: T4: Developing 1 2 Animator should draw a bottle labeled as “reagent 3 0.5 N NaOH Instruct the user to discard the previous reagent, now let user pour the reagent:3 into the tray containing the gel and place it in the incubator for 20 minutes at 65' C. Show a clock running 20 minutes Transfer the gel to the tray containing 0.5N NaOH and place it in incubator for 20 minutes at 65' C. 3 4 5

  16. Description of the action Audio Narration (if any)‏ Step 13: T4: Developing 1 2 3 Instruct user to prepare Reagent:4. Place the required materials on the table, like weighing balance, ammonium molybdate bottle, water and measuring cylinder. Let user weigh 1gram of ammonium molybdate, transfer it to beaker, now measure 1Litre of water and add to beaker. Let user pick the beaker and shake it to mix the solution. Reagent:4 produces molybdate ions which penetrate the gel and form phospho-molybdate complex. 4 5

  17. Description of the action Audio Narration Step 13: T4: Developing 1 Transfer the gel to the tray containing Ammonium Molybade solution and allow it incubate for 10 minutes Repeat the step once more. Animator should draw a bottle labeled as “reagent 4 Ammonium Molybate solution Instruct the user to discard the previous reagent, now let user pour the reagent:4 into the tray containing the gel and leave it for 10 minutes Show a clock running 10minutes repeat this step again by preparing reagent:4 once again. 2 3 4 5

  18. Description of the action Audio Narration (if any)‏ Step 14: T4: Developing 1 2 3 Instruct user to prepare Reagent:5. Place the required materials on the table, like weighing balance, ammonium molybdate bottle, nitric acid, water and measuring cylinder. Let user weigh 1g of ammonium molybdate, transfer it to beaker, add 10ml of nitiric acid to beaker, now measure 1L of water and add to beaker. Let user pick the beaker and shake it to mix the solution. Reagent:5 helps to remove free molybdate ions. 4 5

  19. Description of the action Audio Narration Step 14: T4: Developing 1 Transfer the gel to the tray containing Ammonium Molybdate nitric acid solution and allow it incubate for 20 minutes Animator should draw a bottle labeled as “reagent 5 Ammonium Molybdate nitric acid solution Instruct the user to discard the previous reagent, now let user pour the reagent:5 to the tray containing the gel and leave it for 20 minutes Show a clock running 20minutes 2 3 4 5

  20. Description of the action Audio Narration (if any)‏ Step 15: T5: Stopping 1 2 3 Instruct user to prepare Reagent:6. Place the required materials on the table, like weighing balance, methyl bottle, acetic acid, water and measuring cylinder. Let user weigh 5g of amethyl green, transfer it to beaker, add 10ml of acetic acid in 25ml MC to beaker, now measure 1L of water and add to beaker. Let user pick the beaker and shake it to mix the solution. Reagent:6 helps in staining the phospho-molybdate complex. The complex formed takes up the dye in presence of acetic acid. 4 5

  21. Description of the action Audio Narration Step 15: T5: Stopping 1 Transfer the gel to the tray containing methyl green solution and allow it incubate for 20 minutes. gel takes up the stain and protein spots are visible. Take out bottle “reagent 6 methyl Green Solution”. Instruct the user to discard the previous reagent into the waste and let user pour the reagent:6 into the tray containing the gel and leave it for 20 minutes Show a clock running 20minutes 2 3 4 5

  22. Step 16: T5: Stopping 1 2 3 4 5

  23. Description of the action Audio Narration Step 16: 1 T5: Stopping Transfer the gel to the tray containing sulfosalicylic acid and allow it incubate for 15 minutes Animator should draw a bottle labeled as “reagent 1 Sulfo salicylic acid Instruct the user to pour the previous reagent into the waste and pour reagent:1 to the tray containing the gel and leave it for 15 minutes Show a clock running 15 minutes Each step must happen when the user clicks on the hand 2 3 4 5

  24. Description of the action Audio Narration (if any)‏ Step 17: T5: Stopping 1 2 3 Instruct user to prepare Reagent:7. Place the required materials on the table, like acetic acid, water and measuring cylinder. Let user measure 7ml of acetic acid, transfer it to beaker, now measure 1L of water and add to beaker. Let user pick the beaker and shake it to mix the solution. Reagent:7 helps in destaining the gel. 4 5

  25. Description of the action Audio Narration Step 17: T5: Stopping 1 Transfer the gel to the tray containing 7% acetic acid for de-staining and allow it incubate for 15 hours (overnight) Animator should draw a bottle labeled as “7% acetic acid” Instruct the user to pour the reagent to the tray containing the gel and leave it for 15 hours (overnight) Show a clock running 15 hours (overnight) 2 3 4 5

  26. Step 18: T6: Preservation 1 standard molecular band Standard Molecular band Sample band Standard Molecular band Sample band 2 3 4 5

  27. Description of the action Audio Narration Step 18: T6: Preservation 1 Animator should show the gel as in figure like in previous slide. Once the gel image is obtained after staining user can go through future viewing IDD for more information. 2 3 4 5

  28. Button 01 Button 02 Button 03 Slide 5 Slide 6,7 Slide 8 Slide 9 Slide 10-12 Slide 13 Slide 14,15 Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07 Name of the section/stage Animation area In Slide-26: let user interpret the result of the bands (arrow marked) with the bands of standard molecular marker. Instruction: provide some near values for user to select, in according to the sample bands to that of standard molecular band. Interactivity area Instructions/ Working area Credits

  29. Button 01 Button 02 Button 03 Slide 20,21 Slide 16,17 Slide 18,19 Slide 22,23 Slide 24,25 Slide 26,27 Slide 28,29 Tab 08 Tab 09 Tab 10 Tab 11 Tab 12 Tab 13 Tab 14 Name of the section/stage Animation area Interactivity area Instructions/ Working area Credits

  30. Button 01 Button 02 Button 03 Slide 34,35 Slide 30,31 Slide 32,33 Tab 15 Tab 16 Tab 17 Name of the section/stage Animation area Interactivity area Instructions/ Working area Credits

  31. Questionnaire: APPENDIX 1 Question 1 What is Pro Q diamond? a)Glucostain b)Phosphostain c)Protein stain d)DNA stain Question 2 What is reagent1? a)Sulfsalicylic acid b)Nitro salicylic acid c)Ammonium molybdate d)Calcium chloride Question 3 What is the stain used in gel code stain? a)Methyl orange b)Methyl red c)Methyl green d)Methylene blue

  32. Questionnaire: APPENDIX 1 Question 4 Pro-Q diamond and Methyl green stains a)Phospho stains b)Phosphorous in the cell c)Phospho proteins d)Glucoproteins Question 5 Pro-Q diamond stained gel is scanned in visible light gel scanner at a)555nm b)666nm c)777nm d)888nm

  33. APPENDIX 2 Links for further reading • Reference websites: http://www.piercenet.com/browse.cfm?fldID=02051029 Research papers: Molecular probes(2007).Pro-Q diamond phospho-protein blot stain kit.

  34. APPENDIX 3 Summary The method detect phosphoproteins directly in 1-D and 2-D gels. Steps involved are fixing, staining and destaining, no blotting or antibodies required. Detect phosphate groups attached to tyrosine, serine or threonine residues. Signal is linear over three orders of magnitude and correlates with the number of phosphates present.

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