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Buffer Preparation for Western Blot analysis

Buffers are the important reagents which play a vital role in maintaining the integrity, stringency and efficiency of the experiment. These buffer preparation require atmost care to be successful in an experiment. Buffer Preparation for Western Blot analysis. Related LOs: Reagent property

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Buffer Preparation for Western Blot analysis

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  1. Buffers are the important reagents which play a vital role in maintaining the integrity, stringency and efficiency of the experiment. These buffer preparation require atmost care to be successful in an experiment Buffer Preparation for Western Blot analysis • Related LOs: Reagent property > Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-11. Protein quantification, IDD-17. SDS-PAGE. > Future Viewing – IDD-33. Western blot assay • Course Name: Buffer preparation for western blot analysis • 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 buffer preparation • Operate to optimize the pH of the buffer • Assess the troubleshooting steps involved in the experiments. 2 3 4 5

  3. Master Layout 1 Reagents for western blotting (Slide: 5-7) 2 Buffer preparation (Slide:8-11, 14, 16) Adjusting the pH (Slide:12, 13, 15,18) 3 Adjusting the volume (Slide:17) 4 Use for other experiemnts (Slide:18) 5

  4. Definitions and Keywords 1 1. Transfer Buffer: Transfer buffer contains glycine, Tris Base and methanol. Buffer provides wet transfer condition during electro-blotting of proteins from gel to membrane. This wet transfer is recommended for large proteins and avoids drying of the membrane. 2. Alkaline phosphatase buffer : Alkaline phosphatase buffer contains Nacl,Mgcl2 and tris base. 3. Fixing solution: fixing solution contains methanol, glacial acetic acid and deionized water. 4. De-staining solution: De-staining solution containing sodium acetate and acetonitrile mixture for removing the excess stain from the gels. 2 3 4 5

  5. Step 1: T1:Reagents for western blotting 1 2 Beaker Magnetic bead 3 Description of the action Audio Narration (if any)‏ 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 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 4 magnetic stirrer helps for proper distribution of solute into the solution at faster rate. Video file: Magnetic stirrer 5

  6. Audio Narration ‏ Description of the action The measuring cylinder need to be used to makeup the final required volume. Step 1: T1:Reagents for western blotting 1 2 1000 500 250 3 100 • The animator should draw graduated measuring cylinder as shown in slide with graduation 100ml, 250 ml,500ml,1000ml. The user should click on the appropriate cylinder for usage. 4 5

  7. Step 1: T1:Reagents for western blotting 1 2 3 Audio Narration (if any)‏ Description of the action Show a measuring balance, with display, ON, OFF and TARE/0 buttons on it. let user ON it, display reading as 0.000g, let user picks up the paper from the rack, makes 1/10 of folding on the sides and places it on the balance. Now the display reading changes to 0.003g. Instruct user to TARE the reading. And animate to click the tare button. Once user clicks it, reading must show ”0” When measuring with paper, the weight of the paper need to be tarred from actual reading. 4 5 Video file: Balancing

  8. Step 2: T2: Buffer preparation 1 Tris base Glycine 2 methanol Description of the action Audio Narration 3 Let user pick up glycine, tris base, methanol, spatula, measuring cylinder from the rack and keeps it on the table next to balance. Instruct user to weigh 28.8g of glycine, let user tare the balance, user should click on the glycine bottle, uncap it, with help of spatula weigh the required amount on a paper over the balance. Display a gradual increase in reading with quantity addition. if the gram exceeds user should remove some quantity or if it less add the quantity to get the exact required amount. After weighing transfer the quantity to beaker. Weighing accordingly to the experiment requirement. Prepare transfer buffer containing glycine, Tris Base and methanol. Buffer provides wet transfer condition during electro-blotting of proteins from gel to membrane. This wet transfer is recommended for large proteins to avoid drying of the membrane. 4 5

  9. Step 3: T2: Buffer preparation 1 Tris base Glycine 2 methanol Description of the action Audio Narration 3 Animate like the user, taking methanol bottle (on clicking), open the cap, take 1000ml measuring cylinder, and pour to measure 900ml. Let user remove the excess methanol if level crosses 900ml mark. Tranfer it to beaker. Now take the beaker, place it on magnetic stirrer (follow as in slide 5). Animate the powder getting into the solution. Now transfer the beaker solution to 1000ml measuring cylinder and click on the methanol and add to the cylinder until the volume shows 1000ml. If your are using nitrocellulose, buffer must be prepared using methanol. if using PVDF, distilled water can be used to prepare the buffer. Methanol is needed at the time of electroblotting to activate the PVDF. Now to the beaker, add methanol 4 5

  10. Step 4: T2: Buffer preparation 1 Tween 20 Tris base Nacl 2 Kcl Description of the action Audio Narration 3 Prepare TBST buffer can be used as destain solution and also can be used for washing the membrane. Let user takes out Nacl, Kcl,Tris Base, tween 20 from the rack and keep it next to balance. Instruct user to weigh 8g of Nacl, 0.2g of Kcl, 3g of tris base and 600ul of tween 20. let user pick the bottle, uncap it, weigh the required amount with help of spatula on a paper over the balance. Display a gradual increase in reading with quantity addition. if the gram exceeds user should remove some quantity or if it less add to get the required amount. After weighing transfer the quantity to beaker. Now take out 1000ul pippette, set it for 600ul, take out tween20 bottle, uncap it, pipette and transfer 600ul into the beaker. All events must happen when the user clicks on the hand. 4 5

  11. Step 5: T2: Buffer preparation 1 Tween 20 Tris base Nacl 2 Kcl Description of the action Audio Narration 3 The pH of the TBST buffer need to set to 7.6 for better result. Now instruct the user to take methanol bottle, open the cap, take 1000ml measuring cylinder, measure 900ml. Let user remove the excess methanol if level crosses 900ml mark. Transfer it to beaker. Now take the beaker, shake it to make a proper mix as shown in slide:11. Animate the powder getting into the solution. Now set the pH to 7.6 by using pH meter. 4 5

  12. Step 6: T3: Adjusting the pH 1 2 STD 1 STD 2 3 Audio Narration Description of the action Before the pH reading, pH instrument need to be calibrated with standards. Once with STD 1 at pH 4 and with STD 2 at pH 9. Display standard pH bottles and pH instrument and deionized water, discard placed on a table. Instruct user to caliberate the instrument. Let user ON the instrument. Initially for the pH rod is dipped in water, when user clicks on read button, display must show a reading “7”. Now show like taking out the rod and washing it with deionized-water, let user cleans the rod with tissue. Now pick the STD 1 , uncap it, dip the cleaned rod into the solution, user must click read button with display showing “4”. now clean the rod and repeat the step to note down the reading for STD 2 and now the display should show “9” 4 5 Video file: pH meter

  13. Step 7: T3: Adjusting the pH 1 2 NaOH HCl 3 Audio Narration Description of the action Instruct user to set the pH for TBST pH at 7.6. Now take the TBST bottle, uncap it, dip the cleaned pH rod into the solution. User need to click on read button. Initially display must show a reading 6. now instruct user to add NaOH to adjust the pH. Now allow the user to click on NaOH bottle so that drops of NaOH should be added with filler, user need to mix the solution with glass rod, click on read button and the reading should anywhere near 6.1- 6.3. let user keeps adding the NaOH drop till the pH display shows 7.6 and later transfer the beaker solution to 1000ml measuring cylinder to makeup the volume to 1000ml by clicking on methanol and adding it to that. All action should happen when the user clicks the hand Prepare TBST buffer of pH 7.6 used for washing. 4 5

  14. Step 8: T2: Buffer preparation 1 Tris base Nacl 2 Mgcl2 Description of the action Audio Narration 3 Prepare Alkaline phosphatase buffer containing Nacl,Mgcl2 and tris base, which helps in visualization of bands. Pop-up note “Prepare Alkaline phosphatase buffer “ Show the bottles labeled as Nacl, Mgcl2,Tris Base. The user should click on the required reagent bottle and spatula for weighing. Instruct user to weigh 0.585g of Nacl and 0.102 g of Mgcl2 and 1.11g of Tris and, let user pick the bottle, uncap it, with help of spatula weigh the required amount on a paper over the balance. if the gram exceeds he should remove some quantity or if it low add to get required amount. instruct user to click on methanol and pour in the measuring cylinder and measure 500ml methanol in the measuring and mixing it as shown in slide 5 and Now transfer the beaker solution to 1000ml measuring cylinder and click on the methanol and add to the cylinder until the volume shows 1000ml 4 5

  15. Step 9: T3: Adjusting the pH 1 2 NaOH 3 HCl Audio Narration Description of the action Then the beaker labeled as “ALP pH 9.8” has to be taken near pH meter and allow the user to dip ph rod in the solution. Animate like the user switching on the pH meter. The meter should show pH 8 in the display and instruct user to add NaOH. Now allow the user to click on NaOH so that drops of NaOH should be added in fillers and the reading should increase like.8.1,8.3 and then 8.8,9.2,9.3 and 9..8(desired pH). (follow the instruction like in slide:11 & 12) Adjust the pH of the alakaline phosphatase buffer to 9.8 4 5

  16. Description of the action/ interactivity Audio Narration (if any)‏ Pop-up note “ Prepare Fixing solution “ Show the bottles labeled as ethanol, glacial acetic acid, measuring cylinder and water. Instruct user to measure 10ml of methanol, 7 ml glacial acetic acid , amount to be displayed displayed, click on bottle, pick each bottle to pour the required amount in measuring cylinder. 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 make up the final volume to 100ml with deionized water and transfer the solution into the fixing solution bottle. Step 10: T2: Buffer preparation 1 FIXING SOLUTION 2 WATER 3 Fixing solution helps to dissolve the salts of silver or dyes on plates or paper, on which photographic images need to be developed.  4 5

  17. Step 11: T4: Adjusting the volume 1 Sodium Acetate 2 Description of the action Audio Narration 3 Prepare destaining solution containing sodium acetate and acetonitrile, which helps to remove access stain from the paper or gel. Pop-up note “Prepare destaining solution “ Show the bottles labeled as Sodium acetate and acetonitrile. The user should click on the required reagent bottle and spatula for weighing. Instruct user to weigh 8.2g of Sodium acetate and, let user pick the bottle, uncap it, with help of spatula weigh the required amount on a paper over the balance. if the gram exceeds he should remove some quantity or if it low add to get required amount. Instruct the user to dissolve the weighed amount by adding 80ml of water and 20 ml methanol by measuring using the measuring cylinder and giving a brief spin to dissolve it as in slide 5 4 5

  18. Step 12: T5: Use for other experiemnts 1 2 NaOH 3 HCl Audio Narration Description of the action Then the beaker containing(labeled as “Destaining solution pH 4”) has to be taken near pH meter and allow the user to dip ph rod in the solution. Animate like the user switching on the pH meter. The meter should show pH 5 in the display and instruct user to add Hcl. Now allow the user to click on Hcl so that drops of Hcl should be added in fillers and the reading should decrease like 4.8,4.6,4.4,4.2,4) Adjust the pH of the Destaining solution to 4. now prepared reagents, buffers and solutions can be used in the future viewing IDD. 4 5

  19. Slide Slide:8-11, 14, 16 Button 01 Button 02 Button 03 Slide 5-7 Slide 17 Slide 12,13 Slide 18 Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Name of the section/stage Animation area Interactivity area Slide 5 Show like the user forget to add the magnetic bead for mixing in the magnetic stirrer and show like the solution remains unmixed Instruction: Instruct the user to add the magnetic bead to the beaker and switching on the instrument and show the solution mixes well Instructions/ Working area Credits

  20. Questionnaire: APPENDIX 1 Question 1: What is the pH of destaining solution? 5.5 4.5 4 6 Question 2: What is the use of transfer buffer? To transfer large proteins from the membrane to gel and to wet the gel To transfer large proteins from the membrane to gel and to dry the gel To help in the tranfer of high molecular proteins and to keep the membrane wet To wet the instrument Question 3: What is the need of methanol? To wet the membrane To activate the membrane To dry the membrane Prevent the membrane from tearing during handling

  21. Questionnaire: APPENDIX 1 Question 4: Hcl is used in pH measurement in order to Increase the pH of the solution Decrease the pH of the solution To chlorinate the solution Alter the composition of solution NaOH is used in pH measurement in order to Increase the pH of the solution Decrease the pH of the solution To chlorinate the solution Alter the composition of solution

  22. APPENDIX 2 Links for further reading Books: Biochemistry by Stryer et al., 5th edition Biochemistry by A.L.Lehninger et al., 3rd edition Biochemistry by Voet & Voet, 3rd edition

  23. APPENDIX 3 Summary Buffers need to be prepared with required amount of reagents that can maintain the ionic strength of the solution. pH is important for a buffer preparation. All the reagents prepared is to be properly labeled, prepared fresh for the analysis purpose.

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