Apr 01, 2024
SOP – 2-step protein fractionation (Triton) from fly heads
- 1University of Florida
Protocol Citation: ALFONSO.M.PENA 2024. SOP – 2-step protein fractionation (Triton) from fly heads. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbz151gpk/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: 97609
Keywords: ASAPCRN
Funders Acknowledgement:
Martin Pena, Alfonso
Grant ID: NIH P40OD018537
Abstract
SOP – 2-step protein fractionation (Triton) from fly heads
Materials
Vortex
Tabletop centrifuge
Biomasher II Tube/Pest, Sterile – KIMBLE (Cat #9749625002) – for homogenization.
Biomasher II homogenization pestles
ThermoScientific 1.5ml microtube WX und MX (Cat #314352H01) – For ultracentrifugation.
Pipettes, pipette tips
1.5 mL Eppendorf tubes
Waste bucket and bag
Gloves
Thin marker
Dry ice
Ice
Metal tube rack for ice
Reagents Needed:
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
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.
Protocol references