Apr 01, 2024
SOP – 3-step protein fractionation from fly heads
- 1University of Florida
Protocol Citation: ALFONSO.M.PENA 2024. SOP – 3-step protein fractionation from fly heads. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8emolmk/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: April 01, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 97610
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020527
Abstract
SOP – 3-step protein fractionation from fly heads
Reagents Needed:
Reagents Needed:
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.
Protocol:
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 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.