ProteoExtract Protein Precipitation Kit Protocol (Illustrated)

Prepare Precipitation Agent (Kit components)

To one bottle of

Precipitant 1

(29 mL), add 1.7 mL of Precipitant 2, 3 and 4.

MIX WELL, and label

PRECIPITATION AGENT PA

(34.1 mL solution;

store at - 20°C

).

Prepare Wash Solution

Add 150 mL ethanol to the provided Wash solution (Kit component).

Mix and reconstituted

WASH SOLUTION

can be stored for 1 year at

- 20°C

.

PROCEED FOR PRECIPITATION/CLEANUP

Take equal amounts (as close as possible) of each protein in 200 μL volume Add 800 μL cold

PA

(Precipitation Agent) vortex briefly (10 sec) Incubate at - 20°C for 60 min, OR for 45 min at - 45°C.

Centrifuge at 15,000 rpm for 10 min at 4°C.

Aspirate the pellet First decant solution, then spin down & remove remaining solution with pipette Decant Spin down Remove with tip completely 500 μL cold Wash Solution Wash the PELLET

by tapping

and slight vortexing.

Centrifuge at 15,000 rpm for 2 min at 4°C.

Dissolve the PELLET in buffer of choice (or

LB-TT

)

Repeat 1 time

First decant, then spin down and remove with tip.

Completely aspirate the pellet and air dry the pellet for 5 min (1-DGE) or 1 h (2-DGE).

Supplementary Figure 2 Hori et al.