Dec 01, 2023
Human Brain Sequential Extraction (Tau)
- Michael X. Henderson1
- 1Van Andel Institute
Protocol Citation: Michael X. Henderson 2023. Human Brain Sequential Extraction (Tau). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p9y8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 91707
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-020616
Abstract
This protocol details Human Brain Sequential Extraction (Tau). This protocol is an adaptation of the work of several labs.
Attachments
911-2360.pdf
342KB
Materials
Buffers and Reagents
Base Buffer (1L) Store at 4 °C
| A | B | C | |
| Final Concentration | Stock Buffer | To Add | |
| 10 mM | 0.5 M Tris, pH 7.5 | 20 mL | |
| 0.8 M | 5 M NaCl | 160 mL | |
| 1 mM | 0.5 M EDTA, pH 7.4 | 2 mL | |
| 10% | Sucrose | 100 g | |
| 0.1% | Sarkosyl (25%) | 4 mL | |
| DI water | To 1 L |
25% Sarkosyl (50 mL) Store at Room temperature
Note
Sarkosyl powder should be weighed in a fume hood due to its propensity to drift into the lungs.
| A | B | |
| Compound | To Add | |
| Sarkosyl | 12.5 g | |
| Water | To 50 mL | |
| Rotate overnight | ||
0.5 M PMSF (50 mL) Store at 4 °C
| A | B | |
| Compound | To Add | |
| PMSF | 4.35 g | |
| 100% Ethanol | 50 mL |
1 M DTT (32.5 mL) Aliquot and store at -20 °C
| A | B | |
| Compound | To Add | |
| DTT | 1 g | |
| Water | 6.5 mL | |
| Store in 100 μL aliquots | ||
Reagents
| A | B | C | D | |
| Reagent | Catalog | Vendor | Price | |
| cOmplete Protease Inhibitor | 11873580001 | Millipore Sigma | $434 | |
| Phosphatase Inhibitor Cocktail 3 | P0044-1ML | Millipore Sigma | $90 | |
| Phosphatase Inhibitor Cocktail 2 | P5726 | Millipore Sigma | $96 | |
| PMSF | P7626-25G | Millipore Sigma | $311 | |
| DDT | D0632-5G | Millipore Sigma | $140 | |
| Sarkosyl | L9150-100G | Millipore Sigma | $64 | |
| 27G ½ Needle | ||||
| 19G 1 ½ Needle |
cOmplete™, EDTA-free Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #11873580001
Phosphatase Inhibitor Cocktail 2Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5726 Phosphatase Inhibitor Cocktail 3Merck MilliporeSigma (Sigma-Aldrich)Catalog #P0044
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 PMSFMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626 N-Lauroylsarcosine sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #L9150
1 Day Before Extraction
1 Day Before Extraction
Schematic
Make sure glass 100 or 40 mL homogenizers and pestles (A and B) are cleaned with 70%
ethanol, wrapped in foil, and autoclaved.
Clean out ultracentrifuge tubes and caps with 70% ethanol and dry.
Transfer brain tissue to -20 °C freezer the night before (speeds up thaw).
Ensure there are sufficient protease inhibitor, phosphatase inhibitor, and DTT.
Day 1 - Preparation
Day 1 - Preparation
Turn on the ultracentrifuge (Optima L-100 XP), set the temperature for 4 °C and turn the vacuum on. Make sure Ti-45 and Ti-70 rotors are available. They may be in room 5124.
Fill 2 buckets with wet ice.
Warm PMSF to Room temperature .
Record information about the tissue to be extracted. Note the weight of the dish to be used for
weighing the gray matter.
Fill a 15 mL conical with 10% neutral buffered formalin (NBF) and label with case number.
Day 1 - Extraction
Day 1 - Extraction
Thaw bag(s) of brain tissue On ice .
Move brain to petri dish and weigh it.
Once tissue is thawed, remove meninges and blood vessels. Cut one thin, representative piece
off and transfer it to 10% neutral buffered formalin for fixation and subsequent IHC.
Carefully resect the remaining gray matter from white matter in ice-cold PBS. Transfer gray matter
to a petri dish On ice to be weighed.
Weigh gray matter.
Prepare 30 mL Extraction Buffer per gram gray matter. Add the following to ice-cold Base
Buffer: cOmplete protease inhibitor (1 tab/50 mL ), phosphatase inhibitors (2&3) (1:100), 0.1 millimolar (mM) PMSF (1:5000), 2 millimolar (mM) DDT (1:500).
Add 9 volumes of Extraction Buffer to the homogenizer tube. Cut gray matter into small bits,
and use a buffer to transfer these bits to the tube.
Homogenize with pestle A until the pestle moves easily.
Homogenize with pestle B 3x 25 strokes. Save 200 µL of this as Total Homogenate.
Pour homogenate evenly into Ti-45 tubes (fits ~50 mL /tube). Balance tubes to within 0.1 g .
Spin at 11300 rpm in the Ti-45 (10000 x g ) for 00:10:00 at 4 °C .
10m
Pour supernatant into 50 mL conical by filtering it through a piece of kimwipe folded into two
layers placed in a funnel.
Note
Save 200 µL of this as Sup 1.
Add 9 volumes Extraction Buffer to the centrifuge tube and homogenizer. Transfer pellet in
buffer to homogenizer.
Homogenize with pestle A until the pestle moves easily.
Homogenize with pestle B 3x 25 strokes.
Note
Save 200 µL of this as Pel 1.
Pour homogenate evenly into Ti-45 tubes (fits ~50 mL/tube). Balance tubes to within 0.1 g .
Spin at 11300 rpm in the Ti-45 (10000 x g ) for 00:10:00 at 4 °C .
10m
During the spin, transfer Sup 1 to a beaker with a stir bar. Add sarkosyl to a final 1%.
Pour supernatant into 50 mL conical by filtering it through a piece of kimwipe folded into two
layers placed in a funnel.
Note
Save 200 µL of this as Sup 2.
Combine Sup 1 and Sup 2 in a beaker with a stir bar. Add sarkosyl to a final 1% concentration
(1/27).
Note
Save 200 µL of this as Total Sup.
Stir Total Sup for 1- 01:30:00 at Room temperature .
1h 30m
Add the Extraction Buffer to the centrifuge tube and transfer the pellet to the homogenizer. You can use the same vol as Sup 2 for homogenization.
Note
- Save 200 µL equivalent of this as Pel 2.
- If you used a smaller volume, correct this by diluting the sample.
Add Total Sup to Ti-45 tubes. Balance tubes to within 0.1 g . Spin at 40000 rpm in the Ti-45 (125000 x g ) for 01:15:00 at 4 °C .
1h 15m
Pipet out supernatant into a beaker. Myelin will have floated. Remove this with a pipette.
Note
- Save 200 µL of this as Sark Sup.
- At this point, the pellet should be tight, sticky, and red-brown in color.
Use the vacuum to aspirate remaining liquid around the Sark Pellet.
Note
Do NOT put tubing into centrifuge tube. You can use multiple connected pipet tips, if needed.
Add 10 mL DPBS to each centrifuge tube to wash the Sark Pellet. Use the vacuum to remove DPBS.
Add 2 mL DPBS to wash Sark Pellet a second time. Use vacuum to remove DPBS.
Add 1 mL DPBS to each tube. Pipette liquid towards the Sark Pellet with a cut P1000 tip until the
pellet has loosened. Use a transfer pipette to transfer the pellet to a Ti-70 centrifuge tube.
Note
This step is tricky. Be careful to not lose the Sark Pellet.
Use a cut P1000 tip to pipette up and down to resuspend the pellet.
Add DPBS into the centrifuge tube to reach maximum volume. Balance tubes to within 0.1 g .
Spin Sark Pel at 50000 rpm in the Ti-70 (18000 x g ) for 00:30:00 at 4 °C .
30m
Save 200 µL of the supernatant as DPBS wash. Remove the remaining supernatant by vacuum.
Add 100 µL DPBS/1 g tissue to the pellet. Use a cut P1000 tip to first loosen the pellet, then
break it up as much as possible without pipetting up and down. Transfer to 1.5 mL tubes.
Note
This is a tricky step as the pellet may stick to the pipette tip.
Vortex briefly and spin in a tabletop centrifuge at 1000 x g for 00:01:00 .
1m
Rock the tube at 4 °C Overnight .
Day 1 - Cleanup
Day 1 - Cleanup
Bleach all homogenizers, tools and tubes, but use only soap for metal lids.
Rinse centrifuge tubes for 00:10:00 after bleaching.
10m
Bleach out ice bucks.
Bleach vacuum flask and rinse out.
Day 2
Day 2
Move fixed brain to leaching buffer and cassette for paraffin embedding.
Spin in a tabletop centrifuge at 1000 x g for 00:01:00 at 4 °C .
1m
Pass the suspension repeatedly through a 27G ½ needle to homogenize.
Note
If the clumps are large, start with the bigger 19G 1 ½ needle. Centrifuge at 1000 x g for 00:01:00 if needed to bring materials to the bottom of the tube.
Transfer the resuspended Sark Pel to an autoclaved 1.5 mL Beckman ultracentrifuge tube.
Add 100 µL DPBS to the centrifuge tube and resuspend well.
Note
Save 30 µL as Sark Pel.
Balance tubes to within 0.1g. Spin in OptimaMAX-TL (room 5124) (TLA100.3 rotor with adaptors) at 45000 rpm (80000 x g ) for 00:30:00 at 4 °C .
30m
Remove supernatant and add 100 µL DPBS/1 g tissue to the pellet.
Vortex to mix. Freeze or continue to process Sark Pellet.
Day 2 or 3 – Further Purification
Day 2 or 3 – Further Purification
1h 0m 2s
Thaw the Sark Pellet.
Sonicate the Sark Pellet with 20x 1 sec pulses with hand sonicator.
Balance tubes to within 0.1g. Spin in OptimaMAX-TL (room 5124) (TLA100.3 rotor with
adaptors) at 45000 rpm (80000 x g ) for 00:30:00 at 4 °C .
30m
Transfer supernatant into another tube as High g Sup using sterile pipette tips.
Resuspend the pellet with 20% of the volume of DPBS (e.g. for 700 µL Sup, add 140 µL ).
Sonicate High g Pellet for 90-120x 00:00:01 pulses with hand sonicator On ice to break up the pellet.
Note
Save 10 µL as High g Pel.
1s
Transfer the resuspended High g Pellet to a 1.5 mL tube. Spin in tabletop centrifuge at 10000 x g for 00:30:00 at 4 °C .
30m
Transfer supernatant using a sterile pipette tip to a 1.5 mL tube.
Note
- Save 20 µL as Low g Sup.
- This is the final supernatant that CONTAINS ENRICHED PATHOLOGICAL TAU.
Add an equal volume of DPBS to the pellet and sonicate 30x 00:00:01 pulses with hand sonicator to resuspend the pellet.
Note
Save 200 µL of the pellet as Log g Pel.
1s