SOP – 3-step protein fractionation from fly heads

Protease and Phosphatase Inhibitors (cOmplete 04 693 124 001, PhosphoSTOP 04 906 837 001)

Urea – Fisher Bioreagents #BP169-500

SDS – Fisher Bioreagents #BP166-500

RIPA Buffer (PierceTM, Thermo Scientific 89900)

NP-40 (Nonidet® P 40 Substitute, Sigma, Cat #74385)

A	B	C
Stock Solutions	Formula	Storage
TBS	20 mM Tris Base, 150mM NaCl, pH 7.6	4C
20% SDS	10g SDS in 50ml of MilliQ	Room Temperature; tends to gel up, in which case you have to heat it up on a hot plate
RIPA Buffer	PierceTM, Thermo Scientific 89900	4C
10X PhosphoStop	1ml of MilliQ; 1 tablet: PhosphoStop	-20C
10X cOmplete	1ml of MilliQ; 1 tablet: cOmpleteMini	-20C

A	B
TBS	800uL TBS, 100uL 10X cOmplete, 100uL 10X PhosphoStop
5% SDS	550ul TBS, 100ul 10X cOmplete, 100ul 10X PhosphoStop, 250ul 20% SDS
RIPA	800uL RIPA Buffer, 100uL 10X cOmplete, 100uL 10X PhosphoStop
1% NP-40	790uL TBS, 100uL 10X cOmplete, 100uL 10X PhosphoStop, 10uL NP-40
8M Urea/5% SDS	300ul TBS, 100ul 10X cOmplete, 100ul 10X PhosphoStop, 250ul 20% SDS
	Add 0.480.4g Urea wait to dissolve then bring to 1ml with TBS

Working solutions (w/ PPIs) Make and use the day of the extraction, keep on ice. Formulation per 1ml.

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 TBS 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 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 (TBS-soluble fraction).

Wash the pellet by adding 50uL of TBS with PPI and centrifuging at 100,000 g for 15 min at 4°C. Discard the supernatant.

Homogenize pellet with 30uL (1.5ul/fly head) of solvent with PPI with hand-held homogenizer.

Centrifuge at 100,000 g for 30 min at 4°C and save supernatant into new labeled collection Eppendorf (_-soluble fraction).

Wash the pellet by adding solvent 2 with PPI and centrifuging at 100,000 g for 15 min at 4°C. Discard the supernatant.

Resuspend pellet in 30ul (1.5ul/fly head) in 8M urea/5% SDS buffer with PPI by sonicating with 3 one-second pulses 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.