Kits de tripsina SMART Digest™

Yes, we recommend the following protocol, which can be optimized as needed:
1. Rinse a Fisher Scientific Vivacon 500, 30kDa MWCO filter (Cat. No. 14558347) to remove trace amounts of glycerin using a fill volume of water. Decant the filtrate and collection vessel. The filter is now ready for use. If you do not want to use the Vivacon filter immediately, then store the pre-rinsed device in the refrigerator with water or buffer covering the membrane surface. Do not allow the membrane to dry out.
2. Dilute sample water to a total volume of 150 µL on the filter.
3. Centrifuge 14,000 RPM for 15 minutes and discard the flow-through.
4. Add 150 µL water.
5. Centrifuge 14,000 RPM for 15 minutes and discard the flow-through.
6. Add 200 µL of SMART Digest buffer.
7. Change the collection vial and add 5 µL of the SMART Digest soluble enzyme to the filter.
8. Parafilm the filters to prevent evaporation of sample during digestion. Incubate the units in a heated bath at 70°C for 60 - 90 minutes.
9. Optional: As needed, add DTT (3 µL of 50 mM DTT), heat at 37°C and incubate for 30 minutes. Alkylate as needed (using 3 µL of 150 mM alkylating reagent).
10. After incubation, add 2 µL of 1% trifluoroacetic acid to the filter. Mix for 1 minute.
11. Centrifuge the filter units at 14,000 RPM for 10 min.
12. As needed, measure protein concentration using a Thermo Scientific NanoDrop Spectrophotometer, or similar procedure.

To automate the SMART Digest Soluble Trypsin Kit workflow, we recommend using deepwell plates with Sepra Seals, or PCR plates with iron-on seals in combination with an automated liquid handling platform. Any heating unit with a heated lid, capable of providing uniform heating at 70°C may be used.

The kit is compatible with up to 1M urea, but incompatible with most concentrations of guanidine.

The kit is compatible with many surfactants including OGS, CHAPS, Nonidet, and NP-40.

Thus far, studies have shown that by accelerating the reaction and minimizing the reaction time, fewer chemical modifications are observed in comparison to protocols utilizing elongated digestion times at lower temperatures. For more details, refer to the application note titled 'SMART Digest Compared to Classic In-Solution Digestion of Rituximab for In-Depth Peptide Mapping Characterization'.