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Resolubilization of Intact Protein Protocol

PREPARATION NOTES
Spin speeds are based on a standard benchtop microcentrifuge with 24 x 1.5/2.0 mL rotor.
Times provided are guidelines only.
If more than a few microliters of liquid remains in the Filtration Cartridge after any spin, return it to the centrifuge and repeat the spin, or consider increasing the spin speed. 3000 ×g (6000 rpm) is recommended for subsequent spins and the ProTrap XG has been tested up to 9000 ×g (10,000 rpm).
The capacity of the ProTrap XG is 250 µg of protein.

MATERIALS REQUIRED
All chemicals and reagents should be ACS grade/HPLC grade or better.
80% Formic acid in water (chilled to –20°C)
Chilled water

RESOLUBILIZATION OF INTACT PROTEIN

  • This procedure applies to samples following solvent precipitation in the Protein Precipitation in Acetone Protocol provided.
  • Once the sample has been precipitated using the provided protocol, with the Plug still attached to the base of the Filtration Cartridge, add 150 µL of cold (-20°C) 80% formic acid in water.
  • Cap the Filtration Cartridge, place in freezer for 10 minutes, then vortex or sonicate for 1 minute.
  • Add 350 µL chilled water; cap and vortex to mix the solvent.
  • Intact proteins may be directly recovered in a clean Microcentrifuge Tube, centrifuging at 350 ×g (2000 rpm) ×5 minutes.
  • Resolubilized proteins may also be subject to SPE using the provided SPE Cartridge and the SPE Protein/Peptide Clean-Up Protocol.

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