ProtoGel Sample Prep Kit

Protocol

Protocol:

1. Add 5 μL of Reagent A for every 100 μL of sample in a microcentrifuge tube and mix well.
2. Add 10 μL of Reagent B for every 100 μL of sample and mix.
3. Incubate for 20 minutes at room temperature, inverting the tube occasionally to promote mixing.
4. Collect complex by centrifugation at 12,000 x g and remove supernatant. The large white pellet contains the precipitant complex and the protein.
5. Add 1 mL acetone and mix well to ensure it completely dissolves the complex. Vortexing is generally sufficient but pipetting up and down may be necessary. There should be no clumps. Depending on the protein concentration the solution will be clear to hazy.
6. Collect proteins by centrifugation at 12,000 x g for 10 minutes. Remove the acetone supernatant. The purified protein pellet will be small and nearly invisible for amounts less than 1 μg.
7. Wash protein pellet at least twice by suspending the pellet in 70% ethanol and collecting proteins by centrifugation. NOTE: These washes are critical to the purity of the recovered protein.
8. Air-dry pellet, mix with Protein Loading Buffer Blue 2X and deionized water to desired volume, heat to 95 ºC for two minutes and load onto SDS-PAGE gel.

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