Buffer Preparation for Western Blot analysis Buffers are

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 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-Non. Commercial-Share. Alike 2. 5 India license

1 Learning objectives • 2 3 4 5 After interacting with this Learning Object, the learner will be able to: 1. Define buffer preparation 2. Operate to optimize the p. H of the buffer 3. Assess the troubleshooting steps involved in the experiments.

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

1 2 Definitions and Keywords 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, Mgcl 2 and tris base. 3. Fixing solution: fixing solution contains methanol, glacial acetic acid and deionized water. 3 4 5 4. De-staining solution: De-staining solution containing sodium acetate and acetonitrile mixture for removing the excess stain from the gels.

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

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

1 Step 1: T 1: Reagents for western blotting 2 3 4 5 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. 000 g, 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. 003 g. Instruct user to TARE the reading. And animate to click the tare button. Once user clicks it, reading must show ” 0” Audio Narration (if any) When measuring with paper, the weight of the paper need to be tarred from actual reading. Video file: Balancing

1 2 Step 2: T 2: Buffer preparation Tris base Glycine methanol 3 4 5 Description of the action 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. 8 g 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. Audio Narration 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.

1 2 Step 3: T 2: Buffer preparation Tris base Glycine methanol 3 4 5 Description of the action Animate like the user, taking methanol bottle (on clicking), open the cap, take 1000 ml measuring Now to the beaker, add cylinder, and pour to measure 900 ml. Let user methanol remove the excess methanol if level crosses 900 ml 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 1000 ml measuring cylinder and click on the methanol and add to the cylinder until the volume shows 1000 ml. Audio Narration 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.

1 Step 4: T 2: Buffer preparation Tween 20 2 Tris base Nacl Kcl 3 4 5 Description of the action Let user takes out Nacl, Kcl, Tris Base, tween 20 from the rack and keep it next to balance. Instruct user to weigh 8 g of Nacl, 0. 2 g of Kcl, 3 g of tris base and 600 ul 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 1000 ul pippette, set it for 600 ul, take out tween 20 bottle, uncap it, pipette and transfer 600 ul into the beaker. All events must happen when the user clicks on the hand. Audio Narration Prepare TBST buffer can be used as destain solution and also can be used for washing the membrane.

1 Step 5: T 2: Buffer preparation Tween 20 2 Tris base Nacl Kcl 3 4 5 Description of the action Now instruct the user to take methanol bottle, open the cap, take 1000 ml measuring cylinder, measure 900 ml. Let user remove the excess methanol if level crosses 900 ml 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 p. H to 7. 6 by using p. H meter. Audio Narration The p. H of the TBST buffer need to set to 7. 6 for better result.

1 Step 6: T 3: Adjusting the p. H 2 3 4 5 STD 1 STD 2 Description of the action Audio Narration Before the p. H reading, p. H Display standard p. H bottles and p. H instrument and deionized water, discard placed on a table. Instruct user to instrument need to be calibrated with standards. caliberate the instrument. Let user ON the instrument. Initially for the p. H rod is dipped in water, when user clicks Once with STD 1 at p. H 4 and on read button, display must show a reading “ 7”. Now with STD 2 at p. H 9. 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” Video file: p. H meter

1 Step 7: T 3: Adjusting the p. H 2 Na. OH HCl 3 4 5 Description of the action Instruct user to set the p. H for TBST p. H at 7. 6. Now take the TBST bottle, uncap it, dip the cleaned p. H rod into the solution. User need to click on read button. Initially display must show a reading 6. now instruct user to add Na. OH to adjust the p. H. Now allow the user to click on Na. OH bottle so that drops of Na. OH 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 Na. OH drop till the p. H display shows 7. 6 and later transfer the beaker solution to 1000 ml measuring cylinder to makeup the volume to 1000 ml by clicking on methanol and adding it to that. All action should happen when the user clicks the hand Audio Narration Prepare TBST buffer of p. H 7. 6 used for washing.

1 2 Step 8: T 2: Buffer preparation Tris base Nacl Mgcl 2 3 4 5 Description of the action Pop-up note “Prepare Alkaline phosphatase buffer “ Show the bottles labeled as Nacl, Mgcl 2, Tris Base. The user should click on the required reagent bottle and spatula for weighing. Instruct user to weigh 0. 585 g of Nacl and 0. 102 g of Mgcl 2 and 1. 11 g 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 500 ml methanol in the measuring and mixing it as shown in slide 5 and Now transfer the beaker solution to 1000 ml measuring cylinder and click on the methanol and add to the cylinder until the volume shows 1000 ml Audio Narration Prepare Alkaline phosphatase buffer containing Nacl, Mgcl 2 and tris base, which helps in visualization of bands.

1 Step 9: T 3: Adjusting the p. H 2 3 4 5 Na. OH HCl Description of the action Then the beaker labeled as “ALP p. H 9. 8” has to be taken near p. H meter and allow the user to dip ph rod in the solution. Animate like the user switching on the p. H meter. The meter should show p. H 8 in the display and instruct user to add Na. OH. Now allow the user to click on Na. OH so that drops of Na. OH 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 p. H). (follow the instruction like in slide: 11 & 12) Audio Narration Adjust the p. H of the alakaline phosphatase buffer to 9. 8

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

1 2 3 4 5 Step 11: T 4: Adjusting the volume Sodium Acetate Description of the action 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. 2 g 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 80 ml of water and 20 ml methanol by measuring using the measuring cylinder and giving a brief spin to dissolve it as in slide 5 Audio Narration Prepare destaining solution containing sodium acetate and acetonitrile, which helps to remove access stain from the paper or gel.

1 Step 12: T 5: Use for other experiemnts 2 3 4 5 Na. OH HCl Description of the action Then the beaker containing(labeled as “Destaining solution p. H 4”) has to be taken near p. H meter and allow the user to dip ph rod in the solution. Animate like the user switching on the p. H meter. The meter should show p. H 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) Audio Narration Adjust the p. H of the Destaining solution to 4. now prepared reagents, buffers and solutions can be used in the future viewing IDD.

Slide 57 Slide: 811, 14, 16 Tab 01 Tab 02 Slide 17 Tab 03 Slide 12, 13 Tab 04 Slide 18 Tab 05 Tab 06 Name of the section/stage Interactivity area Animation 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 Button 01 Button 02 Button 03 Instructions/ Working area Credits

APPENDIX 1 Questionnaire: Question 1: What is the p. H of destaining solution? a) b) c) d) 5. 5 4 6 Question 2: What is the use of transfer buffer? a) b) c) d) 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? a) b) c) d) To wet the membrane To activate the membrane To dry the membrane Prevent the membrane from tearing during handling

APPENDIX 1 Questionnaire: Question 4: Hcl is used in p. H measurement in order to a) b) c) d) Increase the p. H of the solution Decrease the p. H of the solution To chlorinate the solution Alter the composition of solution Na. OH is used in p. H measurement in order to a) b) c) d) Increase the p. H of the solution Decrease the p. H of the solution To chlorinate the solution Alter the composition of solution

APPENDIX 2 Links for further reading 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

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