TITLE: α-Synuclein Protein Preparation (Large scale)
AUTHORS: Michael X. Henderon, Lindsay Meyerdirk

[DESCRIPTION]
This protocol details α-synuclein protein preparation in a large scale.

[GUIDELINES]
Adapted from: Xiao Tu, Kelvin Luk, Jonathan Branch, Patrick O’Brien, Dustin Covell, Katelyn Becker, Volpicelli-Daley, Laura A et al. “Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates.” Nature protocols vol. 9,9 (2014): 2135-46. doi:10.1038/nprot.2014.143

[STEPS]
SECTION: Day 1: Transformation
1. Thaw E. coli (BL21(DE3)RIL) and DNA on ice.

SECTION: Day 1: Transformation
2. Mix 1 µL-2 µL DNA (α-synuclein in pRK172 plasmid) with 20 µL competent cells and incubate 5-10 minutes on ice.

SECTION: Day 1: Transformation
3. Heat-shock cells for 45 s in 42 °C water and place back  on ice for 2 min.

 

SECTION: Day 1: Transformation
4. Add 200 µL LB (no antibiotics) and shake at 37 °C for 60 min.  Warm Amp100 plate @ 37 °C.

SECTION: Day 1: Transformation
5. Plate 200 µL on agar bacteria plate with ampicillin and spread with glass cell spreader.

SECTION: Day 1: Transformation
6. Incubate the plate inverted at 37 °C 60 min.

SECTION: Day 2: Making Starter Culture
10. Add 5 µL of 100 mg/mL Ampicillin to 5 mL SOC/TB/LB in a round bottom tube.

SECTION: Day 1: Transformation- Buffer Preparation
7. Make 4 L of TB and 400 mL of 10x phosphate buffer.

SECTION: Day 1: Transformation- Buffer Preparation
8. Add 450 mL of TB + 50 mL 10x phosphate buffer into 8x 2 L flasks. Add 200 mL to a 1L flask.  Cover the tops with foil, and autoclave on the liquid setting. 

SECTION: Day 1: Transformation- Buffer Preparation
9. Make sure to let flasks cool for at least 60 min after they have been autoclaved, or if you’re in a hurry, cool in the cold room for at least 30 min.

SECTION: Day 2: Making Starter Culture
11. Pick a colony with a 20 µL pipet tip and drop the tip into the starter culture.  Swirl around to ensure the colony detaches from the pipet tip.

SECTION: Day 2: Making Starter Culture
12. Shake at 200 rpm at 37 °C 30 min. 

SECTION: Day 2: Making Starter Culture
13. Add 0.5 mL of the starter culture to each500 mL flask of TB/phosphate buffer and allow to shake in the incubator @ 200 rpm for 30 min without antibiotic.

SECTION: Day 2: Making Starter Culture
14. After 30 min, add 0.5 mL ampicillin (100 mg/mL) and grow 1440 min at 37°C. Expression will occur without induction 30 min. With such a small starter culture, the goal is for at least 1440 min growth.

SECTION: Day 3: Protein Isolation
15. Transfer bacteria cultures to large centrifuge bottles (provided by glassware), make sure the bottles are balanced and centrifuge for 20 min at 5000 x g  and 4 °C (rotor F10S).

SECTION: Day 3: Protein Isolation
15.1. Pour out the supernatant after each spin and keep adding more of the culture to the bottles (balanced) until all culture is centrifuged.
SECTION: Day 3: Protein Isolation
16. 
 

If so, scrape out the pellets and transfer to a labeled 50 mLconical and store at -20 °C. Otherwise:

SECTION: Day 3: Protein Isolation
17. Pour off the supernatant and carefully scrape out pellet with a spatula into a 500 ml beaker. Re-suspend pellets using cold High Salt Buffer. Use 15 mL of the buffer to re-suspend the pellet, pour off into the beaker, and then use 5 mL of the buffer to rinse out the bottle. About 50 mL of High Salt Buffer per liter of cell culture will be used (total after the next step). 

SECTION: Day 3: Protein Isolation
18. Break down pellets in beaker with manual agitation. Pour roughly 40 mL at a time into a 40 mL dounce homogenizer.

SECTION: Day 3: Protein Isolation
19. Homogenize with pestle A (>15 times), making sure that you have broken up all clumps.  Repeat with remaining bacteria until all is homogenized and poured into a small plastic beaker.
SECTION: Day 3: Protein Isolation
19.1. Rinse the homogenizer with the high salt buffer 2-3 times to get the remaining clumps and cell lysate out of the homogenizer
SECTION: Day 3: Protein Isolation
20. Heat electric deep fryer on high heat until water is rapidly boiling, being sure to fill the pot up so that a tube rack can be submerged.
SECTION: Day 3: Protein Isolation
21. Sonicate on ice (in a plastic or metal beaker) at 35% for 80 s total ON time (5 sec ON, 25 sec OFF, 8 min total time).

SECTION: Day 3: Protein Isolation
21.1. You will have to attach the tip to the sonicator, make sure the tip is 1-2cm from the bottom of the cup taking care not to hit/break the tip.
SECTION: Day 3: Protein Isolation
22. Transfer the homogenate to 50 mL conical tubes (~3/4 full).

 
 
SECTION: Day 3: Protein Isolation
23. Make5 L gel filtration dialysis buffer and put in the cold room. 

SECTION: Day 3: Protein Isolation
24. After boiling, bury homogenate tubes in ice and place them in the cold room for at least 30 min or until they feel cold.

SECTION: Day 3: Protein Isolation
25. Centrifuge cold lysate for 20 min at 11800 x g  and 4 °C (rotor F21).

SECTION: Day 3: Protein Isolation
26. OPTIONAL: Take an aliquot of the supernatant. Add 5X sample buffer and run on a 15% gel.
SECTION: Day 3: Protein Isolation
27. Cut dialysis tubing (12-14 kD tubing) and wash in the dialysis buffer to soften the tubing.
SECTION: Day 3: Protein Isolation
28. Pipet supernatant into the tubing, close with clamps (**leave an air bubble to promote floatation of the tubing), put the tubing in dialysis buffer and leave in cold room   on the stirrer (stir slowly!).

SECTION: Day 3: Protein Isolation
29. If you are doing gel filtration tomorrow, prep the ÄKTA (S.1), and start the program for the gel filtration column for loading tomorrow.  (S.2) Lines should be set up as:

A1-Size Exclusion Buffer
A2-Sterile Distilled Water
B1-20% Ethanol
B2-20% Ethanol
SECTION: Day 3: Protein Isolation
30. Clean-up: wash all beakers and tubes with bleach and water. Wash dounce homogenizer with DI water and methanol.
SECTION: Day 5: Gel filtration
31. 
 

Filter the protein through a   filter (1000 mL filter unit due to large surface area). 

SECTION: Day 5: Gel filtration
32. Concentrate down to 2-4 mL/L of cell culture in Amicon Ultra Centrifugal Filter devices at 4000 x g, 4 °C, 15 min/cycle or Sartorius Vivaspin 15R at 5000 x g.
 


SECTION: Day 5: Gel filtration
33. Once all the protein is concentrated down to 10 mL total, filter through a   syringe filter into a 50mL conical tube.

SECTION: Day 5: Gel filtration
34. Load the protein sample onto the column using the A1 inlet, MAX of 13 mL.

SECTION: Day 5: Gel filtration
34.1. When the sample is getting close to the bottom of the red stopper, pause the ÄKTA and add 1-2mL of Gel Filtration Buffer into the 50mL conical so you can pull up more protein, continue the program and stop it when it is close to the bottom again. 

 

SECTION: Day 5: Gel filtration
35. Once the sample is all loaded, continue to the “next breakpoint” to start the elution

a.     Manual->Execute Manual Instructions
     i.     Other-> Next Breakpoint->Insert->Execute
         1.     Select continue
                 a.     The program should start up again
SECTION: Day 5: Gel filtration
36. Make 4 L of Buffer A, and place in the cold room.

SECTION: Day 5: Gel filtration
37. Take 10 µL of the even fractions, mix with 2.5 µL 5x sample buffer run on 15% acrylamide gels (will need 2 to see all the fractions of interest).

SECTION: Day 5: Gel filtration
38. Coomassie stain (or Instant Blue) the gels and collect the clean fractions from the gel filtration, primarily avoiding the high molecular weight protein that has a similar charge to α-synuclein (E.1).
SECTION: Day 5: Gel filtration
39. Cut dialysis tubing (12-14 kD tubing) and wash in Buffer A to soften the tubing.
SECTION: Day 5: Gel filtration
40. Pipette the fractions into the tubing and dialyze the fractions against Buffer A 15 min.

SECTION: Day 5: Gel filtration
41. After the elution, the program will continue automatically to wash the column, and store it in 20% Ethanol.
SECTION: Day 6: MonoQ Column (1)
42. 
 

Connect the MonoQ column between ports 2A (top) and 2B (bottom) (S.1)
SECTION: Day 6: MonoQ Column (1)
43. Switch inputs A1, A2 and B1 and start the MonoQ program (S.3). Lines should be set up as:

A1-Buffer A
A2-Sterile Distilled Water
B1-Buffer B
B2-20% Ethanol
SECTION: Day 6: MonoQ Column (1)
44. Load the sample using A1. While the sample is loading collect the flow through to test so you know you are not losing protein that is not sticking to the column.
SECTION: Day 6: MonoQ Column (1)
44.1. When the sample is getting close to the bottom of the red stopper, pause the ÄKTA and add 3 mL of buffer A into the 50 mL conical so you can pull up more protein, continue the program and stop it when it is close to the bottom again. 

 

SECTION: Day 6: MonoQ Column (1)
45. Once the sample is loaded, continue to the “next breakpoint” to start the wash and anion exchange.
SECTION: Day 6: MonoQ Column (1)
46. Take 10 µL of the even fractions as they come off, mix with 2.5 µL 5x sample buffer run on two 15% acrylamide gels.  If you will run a second day of MonoQ, you can pick the fractions to keep based on the UV trace alone.

SECTION: Day 6: MonoQ Column (1)
47. Coomassie stain the gels and collect the clean fractions from the gel filtration, primarily avoiding proteins that are not α-synuclein (E.2).  This will primarily manifest right before and right after the main peak.
SECTION: Day 6: MonoQ Column (1)
48. Dialyze the fractions against Buffer A (or DPBS if this is pure enough) 15 min.

 

SECTION: Day 6: MonoQ Column (2)
49. Repeat steps 44-48 above, and keep fractions based on coomassie and UV trace and dialyze into DPBS, pH 7.0 20 min.

SECTION: Day 6: MonoQ Column (2)
50. The primary goal is to get rid of truncated α-synuclein. This shows up as a hump on the later end of the a-synuclein peak and can be visualized as truncated forms on the Coomassie-stained gel (E.3).
SECTION: Day 7: Concentrate and Aliquot
51. Concentrate down to approximately 2 mL of α-synuclein or molar equivalent in Amicon Ultra Centrifugal Filter devices at 4000 x g, 4 °C, 10 min/cycle. 

 

SECTION: Day 7: Concentrate and Aliquot
51.1. Measure the protein concentration by a BCA assay with sample dilutions of 1:10, 1:20, 1:40, 1:60, 1:80, 1:160, 1:320, 1:640.
SECTION: Day 7: Concentrate and Aliquot
51.3. Take the average of the concentration estimations for which values are in the linear range of the BSA curve.

 

SECTION: Day 7: Concentrate and Aliquot
51.2. Load only 10 µL of each sample into the BSA assay. 

SECTION: Day 7: Concentrate and Aliquot
52. Filter the protein through a   filter. Since this is a small volume, the small blue syringe filters are ideal to minimize sample loss.

SECTION: Day 7: Concentrate and Aliquot
53. Aliquot the protein into tubes with the protein type, concentration, date and initials.
SECTION: Day 7: Concentrate and Aliquot
54. Freeze at -80 °C (monomer) or proceed with aggregation (fibrils).

SECTION: Day 7: Concentrate and Aliquot
55. For aggregation, aliquot the protein out into low binding tubes. 

 

SECTION: Day 7: Concentrate and Aliquot
56. Place tubes in a heated shaker at 1000 rpm at 37 °C for 10080 min. 

SECTION: Day 7: Concentrate and Aliquot
57. After 7 days, aliquot the fibrils into small aliquots to avoid freeze thaw cycles, and store in the mid/back of the -80 °C freezer.

SECTION: Examples of Mouse Alpha Synuclein Prep done on 12.2020
59. 
 

 

 

SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to pull the lines through with the new buffer
60. Place all the lines in their corresponding bottles.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to pull the lines through with the new buffer
61. Using the 25 mL Syringe pull 20 mL through the lines at the correct pump location, do this slowly, if you go too quick you can introduce air into the system die to back pressure. To do this:
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to pull the lines through with the new buffer
61.1. Go to Manual->Execute Manual Instructions->Flow path
       a.     Then you can choose Inlet A or Inlet B
             i.     Then choose your flow path (A1 or A2 for A, B1 or B2 for B)
                  a.     Select Insert
            ii.     Hit the Execute button
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to pull the lines through with the new buffer
61.2. You can now pull the liquid out of the corresponding pumps you just turned on. Do this by:

Turning the knob on the outside of the pump and inserting the syringe tip into the opening and slowly pulling back
Make sure to tighten the pump knobs once you have finished pulling the lines

 


SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
62. 
 

Manual->Execute Manual Instructions-Insert the following instructions
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
63. Confirm on the screen that the B2 path is highlighted and flowing over the column to waste at 2 mL/min or slower.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
64. Unscrew the bottom connector from the Q column (20% Ethanol should be dripping out) and cap the bottom.

 

SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
65. Loosen the connector at the top of the column, and hit continue on the program.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
66. Fill the top of the Q column with 20% Ethanol and screw the top cap on.

 

SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the Superdex Column (top to bottom)
67. If you just detached the Q column you can continue on, if the system was off perform the steps above (1.i->1.iii), their should be 20% Ethanol dripping from the top connecting line.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the Superdex Column (top to bottom)
68. Insert the column into the holders on the ÄKTA carefully (bottom should be in line with the bottom of the pumps).
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the Superdex Column (top to bottom)
69. Remove the cap from the top of the column, 20% Ethanol should start dripping out of the tubing from the column. Remove the tubing from 1A location on the ÄKTA (this is the location the 20% Ethanol should be dripping from), connect the column to the port on the machine in a drip to drip fashion. 

 

SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the Superdex Column (top to bottom)
70. Detach the buffer reservoir from the bottom of the superdex column, you should see It dripping out now.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the Superdex Column (top to bottom)
71. Attach the tubing to the line coming from the 1B position on the ÄKTA.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the Superdex Column (top to bottom)
72. Buffer should be flowing and the column should be ready to go.

SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the MonoQ column (top to bottom)
77. If you just detached the Superdex column you can continue on, if the system was off perform the steps above (1.i->1.iii), there should be 20% Ethanol dripping from the top connecting line.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the MonoQ column (top to bottom)
78. Attach the tubing to the port 1A on the machine, when 20% Ethanol starts dripping from the tubing, remove the cap from the Q trap.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the MonoQ column (top to bottom)
79. Fill the top of the Qtrap with Ethanol so that it is bubbled up at the top and attach the connector to the column.

 

SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the MonoQ column (top to bottom)
80. Remove the cap from the bottom of the column and continue to run the program on the computer.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the MonoQ column (top to bottom)
81. Connect the line from port 1B to the column.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Attaching the MonoQ column (top to bottom)
82. The column should now have the 20% Ethanol running over it.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
83. Method settings: 
column selected (HiLoad 26/600 Superdex 200pg)
pressure limit should be 0.5 MPa, 
column volume (CV): 318.557ml
column position: 1
flow rate 2.6 ml/min
control the flow to avoid overpressure should be selected.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
84. System prep:
A2 is selected
Injection valve with capillary loop is selected to be washed
Fraction collector is selected to be washed
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
85. Column Wash:
2.6 ml/min (select use the same flow rate as in methods setting)
A2 is shown 0% B1
Wash is 1 CV sent to waste (~ 320 mL)

SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
86. Equilibration: 
2.6 ml/min (select use the same flow rate as in methods setting)
A1 is shown, 0% B
Equilibrate until 2 CV (~640 mL)

SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
87. Miscellaneous
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
88. Load sample
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
89. Elution

SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
90. Water wash
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
91. Ethanol equilibration B2.
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
92. This method will include the following steps

System prep 
Wash the system with 20 mL volume per position at 10 mL per min from inlet A1. Make sure the column position “by-pass” is selected.

SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
93. Equilibration
5 ml/min wash of column with A1 inlet for 5CV
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
94. Miscellaneous
pauses the system with the message “load sample”
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
95. Load sample
2 ml/min through A1 for 20 CV
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
96. Column wash
2 ml/min wash of column with A1 inlet for 5 CV
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
97. Elution-15%
2 ml/min linear gradient elution from 100% A1 inlet to 15% B1 inlet over 10 CV. 
Collecting 2 mL fractions after UV goes to 25.00mAu

SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
98. Elution-30%
2 ml/min linear gradient elution from 15% B1 to 30% B1 over 10 CV.
Collecting fixed 2 mL fractions starting at 15%

SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
99. Elution-100%
2 ml/min linear gradient from 30% B1 to 100% B1 over 5 CV.
Collecting 2 mL fixed fractions

SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
100. Column wash
5 ml/min 100% B1 inlet over 5 CV
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
101. Equilibration of Column
5 ml/min A1 inlet for 5 CV
SECTION: S.3 Henderson Lab MonoQ Qtrap HP Protocol
102. Ethanol wash
5 ml/min 100% B2 inlet line for 5CV

SECTION: Day 3: Protein Isolation
22.1. Boil the homogenate for 20 min.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
62.1.  i. Pumps
System Flow: 2 ml/min
Pressure control: System Pressure
       a.   INSERT
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
62.2.  ii.     Flow Path
Inlet B-> B2-> INSERT
Column Position-> select 1-> INSERT
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Q column
62.3.  iii. Alarms
Alarm system pressure-> Enabled
High alarm = 0.5 mPA
       a. INSERT
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
73. Manual-> Execute Manual Instructions-Insert the following instructions
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
73.1. i. Pumps
1. System Flow: 2 ml/min
2. Pressure control: System Pressure
              a. INSERT
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
73.3. iii. Alarms
1. Alarm system pressure-> Enabled
2. High alarm = 0.5 mPA
     a. INSERT
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
73.2. ii. Flow Path
1. Inlet B-> B2-> INSERT
2. Column Position->select 1 -> INSERT
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
74. When you see 20% Ethanol dripping from system, you can detach the bottom connector and attach the buffer reservoir, allow the ÄKTA to flow over the column until the reservoir is filled so that the stopper is pass the line.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
76. Carefully remove the column and store it in the bottom of the fridge.
SECTION: Supplemental Information: S.1 Preparing the AKTA Chromatography System - How to change out the Columns - Detaching the Superdex Column (bottom to top)
75. Detach the top of the column, 20% Ethanol should be dripping out, quickly cap the top of the tubing off.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
87.1. Message is selected “load sample”.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
87.2. Pause after message is selected.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
88.1. Select “reset UV monitor”.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
88.2. Select “use the same flow rate as in method setting”.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
88.3. A1 should be shown with 0% B.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
88.4. Equilibrate until “the total volume is 0.2 CV” is selected.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
89.1. Select “use the same flow rate as in method settings”.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
89.2. A1 should be shown.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
89.3. Isocratic elution should be selected Volume: 1.5 CV 0% B.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
89.4. Fractionate: select using fraction collector
Fraction type: Peak Fractionation
Peak Frac settings: 50.00 mAu for both start and end level
Peak fractionation volume 5 mL
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
89.5. Select start fractionation after 0.2 CV.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
90.1. Select use the same flow rate as in method settings.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
90.4. Fractionate: select “in waste”.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
90.3. Wash until: select “the total volume is 2 CV.
SECTION: S.2 Size Exclusion Chromatography Program- No Wash Size Exclusion Asyn Purification Day2
90.2. A2 should be shown as the inlet A.
SECTION: Day 7: Concentrate and Aliquot
58. For quality control the sedimentation assay and thioflavin T assay should be done after they fibrillization (see additional protocols).