1.  Related Los: Dye binding properties > Prior Viewing – IDD-17. SDS-PAGE, IDD-18. Second dimension separation of proteins > Future Viewing – IDD-22. 2D-gel scanning and image Analysis, IDD-26. Spot picking  Course Name: Commassie staining Level(UG/PG):UG  Author(s) : Dinesh Raghu, Vinayak Pachapur  Mentor: Dr. Sanjeeva Srivastava Methodology for the Commassie 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 method to detect low concentration of protein is essential for the analysis

2. Learning objectives After interacting with this learning object, the learner will be able to: 1.Define the preparation steps for staining 2.Identify the mechanism behind the staining 3.Operate the steps involved in getting good proteome profile 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 Staining of gel (Slide:9-10) Destaining of gel (Slide:12) Preparation of staining solution (Slide:8) Fixing of gel (Slide:7) Preparation of Fixing solution (Slide:5-6) Washing of gel (Slide:13-15) Preparation of destaining solution (Slide:11)

4. Definitions and Keywords 5 3 2 4 1 1) Coomassie blue: The staining dye in the staining solution is negatively charged dye that binds to the protein and makes protein visible as a spot in the gel. 2) Staining solution: The solution consists of methanol, acetic acid, water and coomassie blue that stains the proteins. 3) Destaining solution: The solution consists of methanol, acetic acid and water, which helps to remove unwanted stains or excess stains from the gel and helps to retains the stain of proteins.

5. Step 1: Audio Narration ‏ Description of the action T1: Preparation of Fixing solution 5 2 1 4 3 The animator should draw graduated measuring cylinder as shown in slide with graduation 100ml, 250ml, 500ml, 1000ml. Let user take out these cylinder from the rack. 100 250 500 1000 The user should click on the appropriate cylinder for usage, when needed should clean and rinse with little amount of distilled water before use.

6. Step 1: Audio Narration (if any)‏ Description of the action/ interactivity Show the bottles labeled as ethanol, glacial acetic acid, measuring cylinder and water. Instruct user to click on bottle and allow him to pick each bottle to pour the required amount say 75 ml ethanol let user pick 100ml cylinder, 25 ml glacial acetic acid let user pick 50ml cylinder and 150 ml water let user pick 250cylinder and measure one by one. 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. Later transfer the solutions into the “fixing solution” bottle. Prepare fixing solution by measuring 75ml of ethanol, 25 ml glacial acetic acid and make up the volume to 250ml with water. Fixing solution helps protein fixation on to the gel and avoid protein diffusion. FIXING SOLUTION WATER T1: Preparation of Fixing solution 1 2 3 5 4

7. Step 2: T2: Fixation of gel Audio Narration (if any)‏ Description of the action/ interactivity Instruct user to pour fixing solution into the staining tray and allow user to pick the gel, kept on glass plate and transfer only gel into the tray. Now animate user control to take the tray containing gel to be placed on ROCKER. Allow user to set the parameters for the rocker and to start the instrument. Set the rocker for medium speed. Animate see-saw movement for the rocker along with solution movement in the tray. Show a clock running 120 minutes Place the gel in the fixing solution to remove the compounds like SDS, ampholytes that produces background. In the meantime prepare the reagent for the next step. GEL Staining trayRocker 1 2 3 5 4

8. Step 3: Audio Narration ‏ Description of the action T3: Preparation of staining solution 5 2 1 4 3 Animator should draw the bottle labeled as methanol, acetic acid, coomassie blue tablets and water, let user takes these things from the rack and place it on the working bench. The user should click on methanol bottle to measure 500ml in 1000ml measuring cylinder(MC), and measure 70ml of Acetic acid in 100ml MC, measure 430 ml of water in 500ml MC pour all the measured solutions into 1000ml MC to make the volume to 1L. Show the user opens the bottle for commassie tablet and take a tablet (blue color) and put in the paper and draw a hammer so that when the user clicks on the hammer the tablet has to be crushed to powder and add the fine powder made to the above made solution. The colorless solution should turn blue and transfer it to “staining solution” Bottle. Animate user keeping the bottle for magnetic stirrer to dissolve the powder completely. Measure the required quantity of acetic acid, methanol and water and add a coomassie tablet powder for 1 liter of the reagent. Methanol AceticAcid Water coomassie blue tablets

9. Step 4: Audio Narration ‏ Description of the action T4: Staining of gel 5 2 1 4 3 Place the gel in the coomassie staining solution containing methanol,acetic acid and coomaassie blue and keep it in the shaker overnight and cover it to prevent evaporation. In the meantime prepare the reagent for the next step. Staining tray Rocker Pour the staining solution into the tray with user interaction. Instruct user to stop the rocker, pick the tray containing gel from the rocker, place it on table. Now transfer the gel into the tray containing staining solution. In other way discard the fixing solution into discard bottle, by slanting the tray and holding the gel. In some case, tray will have opening at the bottom on sides which can be opend to discard the solution. Now animate user control to take the tray containing gel to be placed on ROCKER. Allow user to set the parameters for the rocker and to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray

10. Step 4: T4: staining of gel Audio Narration (if any)‏ Description of the action/ interactivity Show the tray containing gel, zoom out a little region from the gel to show moving circle in which “Dye” has to be prescribed, which goes and bind to protein molecule to form a protein-dye complex blue in color. The user should click on the tray to get the reaction. Please redraw the figure The dye molecules bind to proteins to form a protein-dye complex. The formation of the complex stabilises the negatively charged anionic form of the dye producing the blue colour. 1 2 3 5 4 protein “Dye” Protein-dye complex Coomassie blue binds to protein through ionic interactions between negatively charged sulfonate group in dye and amine groups in protein and Vander wall attractions as well.

11. Step 5: Audio Narration ‏ Description of the action T5: Preparation of De-staining solution Animator should draw the bottle labeled as methanol, acetic acid and water. The user should click on methanol bottle to measure 500ml in 1000ml measuring cylinder, and measure 70ml of Acetic acid in 100ml measuring cylinder, measure 430 ml of water in 500ml measuring cylinder pour into 1000ml measuring cylinder to make De-satining solution. The solution must look colorless Measure the required quantity of acetic acid, methanol and water for preparing destaining solution which removes unwanted dye stains in the gel. Methanol AceticAcid Water De-stainsolution 1 2 3 5 4

12. Step 6: T6: De-staining of gel Audio Narration (if any)‏ Description of the action/ interactivity Instruct user stop the rocker, pick the gel from tray and transfer it into tray containing de-stain solution. Now animate user control to take the tray containing gel to be placed on ROCKER. Allow user to set the parameters for the rocker to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray. Place the gel in the De-staining solution for 6hours with gentle shaking till the dyes are removed from the gel. Replenish the solution several times until background of the gel is fully destained. Once the protein profile is visible you can stop the rocker for the next step. Rocker DE-STAINING SOLUTION GEL 1 2 3 5 4

13. Step 7: T7: Washing of gel Audio Narration (if any)‏ Description of the action/ interactivity Instruct user stop the rocker, pick the gel from tray and transfer it into tray containing water solution. Now animate user control to take the tray containing gel to be placed on ROCKER. Allow user to set the parameters for the rocker and to start the instrument. Animate see-saw movement for the rocker along with solution movement in the tray. Place the gel in the water solution for 2hours with gentle shaking. Rocker water GEL 1 2 3 5 4

14. Step 7: Audio Narration Description of the action 5 2 1 4 3 The animator should show the gel as in the slide after the staining is done. Gel showing protein sample stained with coommassie blue. In comparison to other staining, this method is medium sensitivity, steady state method, fast, helps for good quantification, inexpensive, very environment friendly, mass spectrometry compatible. T7: Washing of gel

15. Step 7: Audio Narration Description of the action 5 2 1 4 3 Once user have the stained image, user can proceed to scanning, followed by Gel analysis. In some case after staining the gel can be used for spot picking. Please go through following IDD for more information. T7: Washing of gel The animator should show the gel as in the slide after the staining is done.

16. Animation area In slide 14: Animate on the stained image some fine blue points/spots and let user finds out the reason behind it. Instruction: 1.fine blue points/spots: uneven mixing of coomassie powder into the solution. Instruct user to carry out the mixing step again and later carry out de- staining step. 2.fine blue lines/spots: tissue paper residue on the gel. Instruct user to use a cleaned staining tray and carry out de-staining step. Instructions/ Working area Credits Name of the section/stage Interactivity area Tab 02Tab 03Tab 04Tab 05Tab 06Tab 07 Button 01 Button 02 Button 03 Tab 01 Slide 5- 6 Slide 7 Slide 8 Slide 9- 10 Slide 13-15 Slide 11Slide 12

17. Questionnaire: APPENDIX 1 Quiz Question 1: The charge of coomassie dye is a)Negative b)Positive c)Neutral d) Both a&b Answer: a)negative Question 2 The coomassie dye stains the protein as a result of a)Proton transfer b)Hydroxyl ion transfer c) Electron transfer d) None of the above Answer:c) Electron transfer

18. Questionnaire: APPENDIX 1 Question 3 Constituents of staining solution 1)Methanol 2) Acetic acid 3) water 4)coomassie blue 5)blue dye a)1 &2 b) 2&3 c)all the above d)1,2,3,&4 Answer: d)1,2,3&4 Question 4 Constituents of destaining solution 1)Methanol 2) Acetic acid 3) water 4) bromophenol blue 5)blue dye a)1 &2 b) 2&3 c)all the above d)1,2,3 Answer:d)1,2,3

19. Questionnaire: APPENDIX 1 Question 5 Commassie dye detects protein in range of a)Micrograms b)Milligrams c)Nanograms d)Kilograms Answer: a)Micrograms

20. Links for further reading Reference websites: 2DE Tutorials by Angelika Görg : http://www.wzw.tum.de/blm/deg/Angelika Görghttp://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 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. APPENDIX 2

21. Summary APPENDIX 3 Negatively charged coomassie dye bind to the protein and make the protein to be visible as a spot in the gel. Protein spot takes up dye color once it is stained with Commassie stain. The staining technique is less sensitive and it detects protein of microgram concentration. This staining method is medium sensitivity, steady state method, fast, good quantification, inexpensive, very environment friendly, mass spectrometry compatible.