Sequential 3 Step AExtraction: TBS, TBS-X, Guanidine

From Guojun Bu via Pritam Das, Mayo Clinic Florida

Day before: Sign up for centrifuge and pre-cool Beckman TLA-110 and TLA-120 rotors in fridge overnight.

Day of: Before starting get dry ice to keep brains frozen and ice for the extractions.

Step 1: TBS Extraction buffer (TBS containing 1x protease (Sigma P8340) and phosphatase inhibitor (Roche Phosphatase Inhibitor Cocktail Tablets #04906845001)

  1. Weigh frozen hemi-brains or quarterbrain pieces and add coldTBS extraction buffer 1 ml/ 150 mg tissue (wet weight). The volume of buffer you need (in mls) is the weight of brain (in mgs) divided by 150.
  2. Homogenize tissue in ice-cold extraction buffer by sonication (Branson sonifier with smallest immersion probe, set to power level 2 and “remote”)in short pulses to keep samples from overheating and to avoid frothing.

3.Load equal quantities of homogenate (~600 ml for hemi-brain & ~300 for quarter brain) into ultrafuge tubes (Beckman polycarbonate centrifuge tubes, Cat #343778). Centrifuge at 100 000 x g for 30 min at 4º C (43k rpm for TLA-110 rotor, 48k rpm for TLA-120 rotor on Beckman TL-100 Ultracentrifuge)

4. Collect supernatant (TBS soluble fraction); save pellet for performing TBS-Triton X extractions next.

5. Aliquot in 50 ul units prior to freezing @ -80oC. Avoid freeze thaw cycles.

Only perform ELISA/MSD for transgenic mice.

For ELISA, dilute samples 1:1 in EC buffer.

For MSD, dilute samples 1:1 with TBS containing 10% blocking reagent (Blocker A #R93BA-1, MesoScale Discovery), and analyze for total A40 and total A42 using MesoScale Discovery kits #K150FTE and K150FUE, respectively.

Step 2: TBS-X Extraction buffer (TBS with protease and phosphatase inhibitors plus 1% Triton X-100)

  1. Re-suspend pellet in TBS-X using the same volume as in step 1. Use pipette to gently re-suspend pellet (1 ml tip 20-30 times, then 200 ul tip for 20-30 times) and mix gently by rotation at 4º C for 30 min (in fridge).
  2. Centrifuge at 100 000 x g for 30 min at 4º C (43k rpm for TLA-110 rotor, 48k rpm for TLA-120 rotoron Beckman TL-100 Ultracentrifuge).
  3. Collect supernatant (TBS-X soluble fraction); save pellet for performing Guanidine extractions next.
  1. Aliquot in 50 ul units prior to freezing @ -80oC. Avoid freeze thaw cycles.

For ELISA, dilute samples in EC buffer as follows (1:5 for pre-depositing mice and 1:10 for older mice);

For MSD(with 4 mo APP/TTA animals), dilute samples 1:3 with TBS containing 10% blocking reagent

Step 3: Guanidine Extraction buffer (5M Guanidine in 50 mM Tris-HCl)

1.Re-suspend pellet in Guanidine buffer using the same volume as in step 1. Use pipette to gently re-suspend pellet (1 ml tip 20-30 times, then 200 ul tip for 20-30 times) and mix gently by rotation at room temperature overnight.

2.Move sample into Eppendorf tubes

3. Centrifuge for 30 min at 16,000 x g at RT.

4.Collect supernatant (labeled Guanidine extraction fraction) and store @ -80oC. Avoid freeze thaw cycles.

For ELISA, dilute samples in EC buffer as follows (1:5000 for old mice; 1:100 for younger mice?)

For MSD (with 4 mo APP/TTA animals), dilute samples 1:1000 with TBS containing 10% blocking reagent.