SOP – 2-step protein fractionation (Triton) from fly heads
ALFONSO.M.PENA
Abstract
SOP – 2-step protein fractionation (Triton) from fly heads
Steps
Reagents Needed:
Protease and Phosphatase Inhibitors (cOmplete 04 693 124 001, PhosphoSTOP 04 906 837 001)
Triton X-100 (Millipore Sigma, Cat# T9284)
NaF (Sigma-Aldrich, Cat# S7920)
| A | B | C |
|---|---|---|
| Stock Solutions | Formula | Storage |
| TBS | 20 mM Tris Base, 150mM NaCl, pH 7.6 | 4C |
| 10X PhosphoStop | 1ml of MilliQ; 1 tablet: PhosphoStop | -20C |
| 10X cOmplete | 1ml of MilliQ; 1 tablet: cOmpleteMini | -20C |
| A | B |
|---|---|
| 1% TritonX-100 (1mL) | 800uL TBS, 100uL 10X cOmplete, 100uL 10X PhosphoStop, 10ul TritonX-1001% TritonX-100 (1mL) 800uL TBS, 100uL 10X cOmplete, 100uL 10X PhosphoStop, 10ul TritonX-100 0.0008g NaF |
Working solutions (w/ PPIs)Make and use the day of the extraction, keep on ice
Protocol
Collect 20-40 fly heads via Snap Freeze method into a Biomasher tube and transfer tubes to CTRND on dry ice.
Turn on Sorvall ultracentrifuge and set temperature to 4degC, place rotor inside and turn on vacuum to allow internal temperature to set.
Add 50uL (2.5uL/fly head) of 1% TritonX-100 with PPI to heads in Biomasher II tube and homogenize for 1.5 min with a hand-held automatic homogenizer and Biomasher pestles.
Briefly spin down in tabletop centrifuge to get the liquid to the bottom of the tube.
Sonicate at 35% with 5 one-second pulses for tissue to break down/dissolve. Skip this step for ‘non-sonicated’ extractions.
Clarify by centrifugation in a tabletop centrifuge (2,100g) for 15 seconds.
Move entire liquid sample into ThermoScientific 1.5ml microtube WX und MX (Cat #314352H01) before centrifuging
Centrifuge the supernatant at 100,000 g (29,900 rpm) for 30 min at 4°C using the chilled rotor.
Remove and save Supernatant into new labeled collection Eppendorf (soluble fraction).
Wash the pellet by adding 50uL of TritonX-100 with PPI and centrifuging at 100,000 g for 15 min at 4°C. Discard the supernatant.
Resuspend pellet in 50ul (2.5ul/fly head) in TritonX-100 with PPI by sonication with 3 one-second pulses or until it is fully resuspended. Label and save (insoluble fraction)
Perform BCA. If strapped on time, samples can wait to be quantified and stored at -80C.
