Protein Digestion Protocol
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.
It is essential that once primed the SPE cartridge is not spun to complete dryness.
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.
All chemicals and reagents should be ACS grade/HPLC grade or better.
8 M urea in 50 mM Tris-HCI pH 8.0 with 20 mM DTT freshly prepared
500 mM Iodoacetamide in 50 mM Tris-HCI pH 8.0 freshly prepared
50 mM Tris-HCI pH 8.0 with 2 mM CaCl2
Trypsin diluted in 50 mM Tris HCI pH 8.0 with 2 mM CaCl2
- This procedure applies to samples following solvent precipitation using the Protein Precipitation in Acetone Protocol provided.
- Once the sample has been precipitated using the provided protocol, with the Plug still attached to the Filtration Cartridge:
- Solubilize and Reduce your sample: Add 50 µL 8 M urea in 50 mM Tris-HCI pH 8.0 with 20 mM DTT. Vortex for 30 seconds and sonicate for 10 minutes. Incubate at 37°C for 1 hour to reduce the disulphide bonds.
- Alkylate: Add 10 µL 500 mM iodoacetamide in 50 mM Tris-HCI pH8.0. Incubate for 15 minutes in the dark at room temperature.
- Dilute: Add 400 µL 50 mM Tris-HCI pH 8.0, 2 mM CaCl2
- Digest: Add Trypsin diluted in 50 mM Tris HCI pH 8.0 2 mM CaCl2 at a ratio of 100:1 protein: enzyme. Incubate in a water bath overnight (16-18 hours).
- Stop and Acidify: Add 4.7 µL of 10% TFA. Ensure pH < 3.5.
- Recover or SPE: Peptides may be recovered by centrifuging after removing Plug and placing the Filtration Cartridge in the provided Microcentrifuge Tube (2500 ×g (5000 rpm) ×5 minutes), OR subject to SPE cleanup using the SPE Protein/Peptide Clean-Up Protocol provided.
Note: If your optimized digestion protocol differs in time, temperature, reducing or alkylating reagent, or concentration of Trypsin, get in touch with our team at firstname.lastname@example.org to confirm that your process can be transferred to the ProTrap XG with no issues.