Resolubilization of Intact Protein Protocol
The following suggested protocol has been optimized using maximum and minimum protein concentrations of 0.5 mg/mL and 0.01 mg/mL respectively and is provided to demonstrate the potential uses of the ProTrap XG. More dilute protein solutions require extra care, connect with our Science Team directly through firstname.lastname@example.org for customized protocols. We’re here to help you implement the ProTrap XG and our base protocols in your workflow.
The ProTrap XG device is optimized to process 50 µg of protein.
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).
All chemicals and reagents should be ACS grade/HPLC grade or better.
80% Formic acid in water (chilled to –20°C)
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 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.