Pipetting scheme for TPA with input amount per well in μ g. Plate 1 shows the dilution series for a control lysate and fi ve samples. Plate 2 shows the pipetting scheme for TPA with a triplicate reaction for each sample. Therefore 2 μ l diluted cell lysate from well A1 ( left plate ) is transferred to wells A1–A3 ( right plate ) and so on 

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Context 1

... control lysate (not treated with apoptotic agent) should always be used as control. This TPA is for relative quantifi cation, not absolute quantifi cation. Also every plate has to contain a “no- protein control” (NPC) to calculate Δ C T . Figure 3 shows the standard confi guration of a 96-well plate used in our lab. This plate contains one control lysate with a fi ve-point serial dilution in triplicates, fi ve sample lysates also with a fi ve-point serial dilution in triplicates, and an NPC with a double triplicate. See Note 6 for details before you begin: 1. Place a 96-well plate (plate 1) on ice to prepare the serial dilution. 2. Dilution sample 1: Dilute 4 μ l 12.5 mg/ml cell lysate in 16 μ l Lysate Dilution Buffer (TaqMan ® Protein Assays Open Kit) in well A1 ( see Fig. 4 for 96-well plate 1). Initial input amount is 5 μ g ( see Note 7 ). 3. Place 12 μ l Lysate Dilution Buffer in wells B1–E1. 4. Transfer 12 μ l cell lysate from well A1 to well B1. 5. Pipette up and down several times to mix the sample. 6. Transfer 12 μ l cell lysate from well B1 to well C1. 7. Pipette up and down several times to mix the sample. 8. Transfer 12 μ l cell lysate from well C1 to well D1. 9. Pipette up and down several times to mix the sample. 10. Transfer 12 μ l cell lysate from well D1 to well E1. 11. Pipette up and down several times to mix the sample. 12. Repeat steps 2 – 11 for every sample. 13. Seal plate 1 with a PCR plate-sealing mat. 14. Centrifuge plate 1 briefl y to spin the liquid to the bottom. 15. Place plate 1 on ice. 16. Place a second 96-well plate (plate 2) on ice ( see Note 8 ). 17. Place thawed Assay Probe Dilution Buffer (TaqMan ® Protein Assays Open Kit), Assay Probe A, and Assay Probe B on ice. Do not vortex Assay Probes. 18. Mix gently 216 μ l Assay Probe Dilution Buffer with 12 μ l Assay Probe A and 12 μ l Assay Probe B in a 0.5 ml tube. 19. Add 2 μ l Assay Probe solution ( step 17 ) to each well of plate 2. 20. Transfer 2 μ l of the serial dilution of plate 1 to the well indicated in Fig. 4 in plate 2. 21. Seal plate 2 with a PCR plate-sealing mat. 22. Centrifuge plate 2 briefl y to spin the liquid to the bottom. 23. Incubate the sealed plate 2 using the following thermal-cycling conditions: Binding: 1 Cycle, 37 °C, 60 min Cooling: 1 Cycle, 4 °C, up to 10 min 24. Place plate 2 on ice and continue immediately. 25. Place thawed Ligase Dilution Buffer, Ligation Reaction Buffer, and DNA-Ligase (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) on ice. 26. Dilute 2 μ l DNA ligase in 998 μ l Ligase Dilution Buffer in a 1 ml tube on ice. 27. Invert the tube twice and place the tube back on ice. 28. Mix 600 μ l Ligation Reaction Buffer with 10,908 μ l nuclease- free water and 12 μ l diluted DNA ligase ( step 25 ) in a 15 ml tube. 29. Invert the tube twice and place the tube back on ice. 30. Place plate 2 ( step 22 ) on ice. 31. Add 96 μ l of the ligation solution ( step 27 ) to each well. 32. Seal the reaction plate with a PCR plate-sealing mat. 33. Centrifuge plate 2 briefl y to spin the liquid to the bottom. 34. Incubate the sealed plate 2 using the following thermal-cycling conditions: Ligation: 1 Cycle, 37 °C, 10 min Cooling: 1 Cycle, 4 °C, up to 10 min 35. Place plate 2 on ice and either continue immediately with the real-time PCR step (go to step 43 ) or perform a protease reaction ( steps 36 – 43 ), if the real-time PCR step should be delayed (up to 3 days at 4 °C or up to 2 weeks at −20 °C). 36. Place thawed PBS and protease (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) on ice. 37. Dilute 4 μ l protease in 396 μ l PBS in a 0.5 ml tube on ice. 38. Pipette up and down two times to dilute protease. 39. Add 2 μ l diluted protease to each well. 40. Seal plate 2 with a PCR plate-sealing mat. 41. Centrifuge plate 2 briefl y to spin the liquid to the bottom. 42. Incubate the sealed plate 2 using the following thermal-cycling conditions: Terminate ligation: 1 Cycle, 37 °C, 10 min Inactivate protease: 1 Cycle, 95 °C, 5 min Cooling: 1 Cycle, 4 °C, hold 43. Place plate 2 on ice. 44. Mix 100 μ l thawed Universal PCR Assay (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) with 1,000 μ l Fast Master Mix (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) in a 1 ml tube on ice. 45. Centrifuge tube briefl y to spin the liquid to the bottom. Place the tube on ice. 46. Place a third 96-well reaction plate (plate 3) ( step 34 or 42 ) on ice. 47. Add 11 μ l PCR mix ( step 43 ) to each well. 48. Transfer 9 μ l of the ligation product ( step 34 ) or the protease- treated ligation product ( step 42 ) to each well. 49. Seal plate 3 with PCR plate-sealing mats. 50. Centrifuge plate 3 briefl y to spin the liquid to the bottom. 51. Incubate the sealed plate 3 using the following real-time PCR conditions ( see Note 5 ): Holding stage: 1 Cycle, 95 °C, 20 s Cycling stage: 40 Cycles, 95 °C, 1 s, and 60 °C, 20 s 52. Save the results of the real-time PCR system software. For analysis see Subheading 3.6.2 . Use a real-time PCR system software and a spreadsheet program. 1. Set the threshold cycle ( C T ) to 0.2 and chose automatic baseline. 2. Export the results from the instrument software to a spreadsheet program. 3. Calculate the average C T values for each biotinylated antibody and each NPC. Each biotinylated antibody and the NPC should have four different values. Average C T value = sum of these four values divided by 4. 4. Calculate the Δ C T values for each biotinylated antibody: Δ C T value = average C T value (NPC) minus average C T value (biotinylated antibody). 5. A biotinylated antibody passed the “forced proximity test” if the Δ C T value is equal or higher than 8.5. If the Δ C T value is lower than 8.5 see Note 9 . Data are analyzed using ProteinAssistTM 1.0 (Applied Biosystems, Foster City, CA), which uses the Δ C T2 method to calculate relative protein expression between untreated controls and treated sample. 1. Use the real-time PCR system software to set the threshold cycle ( C ) to 0.2 and choose automatic baseline ( see Fig. 5 ). 2. Open the ProteinAssistTM software and click “Create Study.” 3. Choose μ g/well for “Input Quantity Unit.” 4. Click “Per Study” for reference use. 5. Click “Experiment Files.” 6. Click “Import” and choose the real-time PCR fi le ( step 52 ). ProteinAssistTM software supports *.csv, *.eds, and *.txt fi les. 7. Import the fi le. 8. Select the imported fi le. 9. Right click a well and select “Edit Well(s).” 10. Assign every well of the control lysate to the task “Reference.” 11. Select for every NPC well the task “NPC.” 12. Select for every sample well the task “Unknown.” 13. Right click a well and select “Edit Well(s).” 14. Assign the correct input amount to every well, for example well A1 “Input Quantity” 5. 15. Click “Analyze” (green button at the top) ( see Note 10 ). 16. Click “Analysis.” 17. The automatic threshold is 2.0. The threshold depends on the expression level of the evaluated protein in the used cell lysates. The optimized threshold for active caspase-3 in primary meningioma cells is 1.0. 18. Choose the appropriate threshold. 19. Click “Analyze” (after every change in the graph the data has to be analyzed again). 20. The analysis of the PCR plate is shown below the graph. 21. Often the automated linear range has to be adjusted manually ( see Fig. 6 ). 22. Select the control lysate (click box before the sample name). 23. The graph only displays the control lysate. 24. Change the linear range if needed ( see Note 11 ). 25. Outliers are marked as an unfi lled triangle. Omit outliers by right clicking the data point in the graph. Select “Omit.” 26. Repeat steps 22 – 25 for every sample. 27. Click “Fold Change.” 28. The graph displays the results as a bar diagram ( see Fig. 7 ). Below the graph the results are detailed ( see Note 12 ...

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... control lysate (not treated with apoptotic agent) should always be used as control. This TPA is for relative quantifi cation, not absolute quantifi cation. Also every plate has to contain a “no- protein control” (NPC) to calculate Δ C T . Figure 3 shows the standard confi guration of a 96-well plate used in our lab. This plate contains one control lysate with a fi ve-point serial dilution in triplicates, fi ve sample lysates also with a fi ve-point serial dilution in triplicates, and an NPC with a double triplicate. See Note 6 for details before you begin: 1. Place a 96-well plate (plate 1) on ice to prepare the serial dilution. 2. Dilution sample 1: Dilute 4 μ l 12.5 mg/ml cell lysate in 16 μ l Lysate Dilution Buffer (TaqMan ® Protein Assays Open Kit) in well A1 ( see Fig. 4 for 96-well plate 1). Initial input amount is 5 μ g ( see Note 7 ). 3. Place 12 μ l Lysate Dilution Buffer in wells B1–E1. 4. Transfer 12 μ l cell lysate from well A1 to well B1. 5. Pipette up and down several times to mix the sample. 6. Transfer 12 μ l cell lysate from well B1 to well C1. 7. Pipette up and down several times to mix the sample. 8. Transfer 12 μ l cell lysate from well C1 to well D1. 9. Pipette up and down several times to mix the sample. 10. Transfer 12 μ l cell lysate from well D1 to well E1. 11. Pipette up and down several times to mix the sample. 12. Repeat steps 2 – 11 for every sample. 13. Seal plate 1 with a PCR plate-sealing mat. 14. Centrifuge plate 1 briefl y to spin the liquid to the bottom. 15. Place plate 1 on ice. 16. Place a second 96-well plate (plate 2) on ice ( see Note 8 ). 17. Place thawed Assay Probe Dilution Buffer (TaqMan ® Protein Assays Open Kit), Assay Probe A, and Assay Probe B on ice. Do not vortex Assay Probes. 18. Mix gently 216 μ l Assay Probe Dilution Buffer with 12 μ l Assay Probe A and 12 μ l Assay Probe B in a 0.5 ml tube. 19. Add 2 μ l Assay Probe solution ( step 17 ) to each well of plate 2. 20. Transfer 2 μ l of the serial dilution of plate 1 to the well indicated in Fig. 4 in plate 2. 21. Seal plate 2 with a PCR plate-sealing mat. 22. Centrifuge plate 2 briefl y to spin the liquid to the bottom. 23. Incubate the sealed plate 2 using the following thermal-cycling conditions: Binding: 1 Cycle, 37 °C, 60 min Cooling: 1 Cycle, 4 °C, up to 10 min 24. Place plate 2 on ice and continue immediately. 25. Place thawed Ligase Dilution Buffer, Ligation Reaction Buffer, and DNA-Ligase (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) on ice. 26. Dilute 2 μ l DNA ligase in 998 μ l Ligase Dilution Buffer in a 1 ml tube on ice. 27. Invert the tube twice and place the tube back on ice. 28. Mix 600 μ l Ligation Reaction Buffer with 10,908 μ l nuclease- free water and 12 μ l diluted DNA ligase ( step 25 ) in a 15 ml tube. 29. Invert the tube twice and place the tube back on ice. 30. Place plate 2 ( step 22 ) on ice. 31. Add 96 μ l of the ligation solution ( step 27 ) to each well. 32. Seal the reaction plate with a PCR plate-sealing mat. 33. Centrifuge plate 2 briefl y to spin the liquid to the bottom. 34. Incubate the sealed plate 2 using the following thermal-cycling conditions: Ligation: 1 Cycle, 37 °C, 10 min Cooling: 1 Cycle, 4 °C, up to 10 min 35. Place plate 2 on ice and either continue immediately with the real-time PCR step (go to step 43 ) or perform a protease reaction ( steps 36 – 43 ), if the real-time PCR step should be delayed (up to 3 days at 4 °C or up to 2 weeks at −20 °C). 36. Place thawed PBS and protease (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) on ice. 37. Dilute 4 μ l protease in 396 μ l PBS in a 0.5 ml tube on ice. 38. Pipette up and down two times to dilute protease. 39. Add 2 μ l diluted protease to each well. 40. Seal plate 2 with a PCR plate-sealing mat. 41. Centrifuge plate 2 briefl y to spin the liquid to the bottom. 42. Incubate the sealed plate 2 using the following thermal-cycling conditions: Terminate ligation: 1 Cycle, 37 °C, 10 min Inactivate protease: 1 Cycle, 95 °C, 5 min Cooling: 1 Cycle, 4 °C, hold 43. Place plate 2 on ice. 44. Mix 100 μ l thawed Universal PCR Assay (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) with 1,000 μ l Fast Master Mix (TaqMan ® Protein Assays Core Reagents Kit with Master Mix) in a 1 ml tube on ice. 45. Centrifuge tube briefl y to spin the liquid to the bottom. Place the tube on ice. 46. Place a third 96-well reaction plate (plate 3) ( step 34 or 42 ) on ice. 47. Add 11 μ l PCR mix ( step 43 ) to each well. 48. Transfer 9 μ l of the ligation product ( step 34 ) or the protease- treated ligation product ( step 42 ) to each well. 49. Seal plate 3 with PCR plate-sealing mats. 50. Centrifuge plate 3 briefl y to spin the liquid to the bottom. 51. Incubate the sealed plate 3 using the following real-time PCR conditions ( see Note 5 ): Holding stage: 1 Cycle, 95 °C, 20 s Cycling stage: 40 Cycles, 95 °C, 1 s, and 60 °C, 20 s 52. Save the results of the real-time PCR system software. For analysis see Subheading 3.6.2 . Use a real-time PCR system software and a spreadsheet program. 1. Set the threshold cycle ( C T ) to 0.2 and chose automatic baseline. 2. Export the results from the instrument software to a spreadsheet program. 3. Calculate the average C T values for each biotinylated antibody and each NPC. Each biotinylated antibody and the NPC should have four different values. Average C T value = sum of these four values divided by 4. 4. Calculate the Δ C T values for each biotinylated antibody: Δ C T value = average C T value (NPC) minus average C T value (biotinylated antibody). 5. A biotinylated antibody passed the “forced proximity test” if the Δ C T value is equal or higher than 8.5. If the Δ C T value is lower than 8.5 see Note 9 . Data are analyzed using ProteinAssistTM 1.0 (Applied Biosystems, Foster City, CA), which uses the Δ C T2 method to calculate relative protein expression between untreated controls and treated sample. 1. Use the real-time PCR system software to set the threshold cycle ( C ) to 0.2 and choose automatic baseline ( see Fig. 5 ). 2. Open the ProteinAssistTM software and click “Create Study.” 3. Choose μ g/well for “Input Quantity Unit.” 4. Click “Per Study” for reference use. 5. Click “Experiment Files.” 6. Click “Import” and choose the real-time PCR fi le ( step 52 ). ProteinAssistTM software supports *.csv, *.eds, and *.txt fi les. 7. Import the fi le. 8. Select the imported fi le. 9. Right click a well and select “Edit Well(s).” 10. Assign every well of the control lysate to the task “Reference.” 11. Select for every NPC well the task “NPC.” 12. Select for every sample well the task “Unknown.” 13. Right click a well and select “Edit Well(s).” 14. Assign the correct input amount to every well, for example well A1 “Input Quantity” 5. 15. Click “Analyze” (green button at the top) ( see Note 10 ). 16. Click “Analysis.” 17. The automatic threshold is 2.0. The threshold depends on the expression level of the evaluated protein in the used cell lysates. The optimized threshold for active caspase-3 in primary meningioma cells is 1.0. 18. Choose the appropriate threshold. 19. Click “Analyze” (after every change in the graph the data has to be analyzed again). 20. The analysis of the PCR plate is shown below the graph. 21. Often the automated linear range has to be adjusted manually ( see Fig. 6 ). 22. Select the control lysate (click box before the sample name). 23. The graph only displays the control lysate. 24. Change the linear range if needed ( see Note 11 ). 25. Outliers are marked as an unfi lled triangle. Omit outliers by right clicking the data point in the graph. Select “Omit.” 26. Repeat steps 22 – 25 for every sample. 27. Click “Fold Change.” 28. The graph displays the results as a bar diagram ( see Fig. 7 ). Below the graph the results are detailed ( see Note 12 ...