Download Top Down Sample Prep Protocol (PDF)
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.
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The ProTrap XG device is optimized to process 50 µg of protein.
For reproducible and maximal protein precipitation, the maximum SDS content in your sample during precipitation should be 1%. If your extraction or lysis buffer contains more than 1% SDS, dilute it with a buffer containing a maximum ionic strength of 300 mM.
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.
5 M NaCl in water
80% Formic Acid in water (chilled to –20°C)
TOP DOWN SAMPLE PREP PROTOCOL
For reproducible and maximal protein precipitation, the maximum SDS content in your sample during precipitation should be 1%. If your extraction or lysis buffer contains more than 1% SDS, dilute it with a buffer containing a maximum ionic strength of 300 mM. For the best experience, your sample should contain at least 50 mM NaCl. If you need to add NaCl, use a 5 M NaCl solution.
- Do not allow the membrane in the Filtration Cartridge to dry out between steps.
- Screw a Plug onto the base of the Filtration Cartridge.
- Transfer 100 µL of the diluted sample protein to the plugged Filtration Cartridge.
- Add 400 µL room temperature acetone.
- Cap the Filtration Cartridge and rock gently, tilting no more than 45°, to combine the solvents.
- Insert the Filtration Cartridge in the Microcentrifuge Tube, allow 30 minutes for the protein to fully aggregate at room temperature.
- With Plug attached, centrifuge at 2500 ×g (5000 rpm) ×2 minutes.
- Remove Filtration Cartridge from the Microcentrifuge Tube, invert, and unscrew the Plug.
- Return the capped Filtration Cartridge to the Microcentrifuge Tube and centrifuge at 500 ×g (2000 rpm) ×3 minutes. Discard the flow through solvent. If any solvent remains in the Filtration Cartridge, re-spin the unit for 2-5 minutes at 3000 ×g (6000 rpm).
- Wash the protein pellet with 400 µL acetone. Immediately centrifuge at 500 ×g (2000 rpm) ×2 minutes. Discard the flow through wash solvent.
- Replace the plug. Chill the pellet in the Filtration Cartridge for 10 minutes at –20°C.
- Add 50 µL of cold (-20°C) 80% formic acid in water. Cap the Filtration Cartridge, place in the freezer for 10 minutes, then sonicate for 1 minute.
- Add 450 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.
If further protein clean-up is desired, resolubilized proteins may also be subject to SPE using the provided OPTIONAL SPE Cartridge and the SPE Protein/Peptide Clean-up Protocol.