Hi,

The below protocol works fine for me. This is just a regular protocol.

Passive Elution of Proteins from Polyacrylamide Gel Pieces:

1. Place excised gel pieces in clean screw-cap culture or microcentrifuge tubes.

2. Add 0.5-1 ml of elution buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM EDTA; pH 7.5) so that the gel pieces are completely immersed.

3. Crush the gel pieces using a clean pestle and incubate in a rotary shaker at 30°C overnight.

4. Centrifuge at 5,000-10,000 × g for 10 minutes and carefully pipette supernatant into a new microcentrifuge tube. An aliquot of the supernatant may be tested for the presence of protein by subjecting it to SDS-PAGE.

If elution volume is a limitation to detect your protein, I suggest you to do a speedvac or concentrate the sample after elution.