Protocol of SPEED

Protein concentration using nanodiamonds prior to SDS-PAGE analysis

  1. Mix protein solution with formic acid to a final concentration of 1% formic acid.
  2. Add nanodiamonds (diamonds/protein = 50/1 w/w) into the solution.
  3. Incubate at room temperature for 5 minutes.
  4. Centrifuge at 12000 rpm for 5 minutes.
  5. Remove (and discard) supernatant.
  6. [Optional] Dry the pellet - in vacuum or just leave the tube in the hood - so that residual formic acid will escape.
  7. Resuspend pellet with 1x SDS sample buffer (if needed, eg. When step 6 is skipped, use 1M Tris pH8.8 to neutralize remaining formic acid).
  8. Roughly sonicate the sample (5 seconds, use water-bath type sonicator).
  9. Incubate at 95oC for 5 minutes.
  10. Apply the sample-diamond mixture to SDS-PAGE analysis (the presence of diamond particles does not interfere with PAGE).

On-diamond trypsin digestion

  1. Mix protein solution with formic acid to a final concentration of 1% formic acid.
  2. Add nanodiamonds (diamonds/protein = 50/1 w/w) into the solution.
  3. Incubate at room temperature for 5 minutes.
  4. Centrifuge at 12000 rpm for 5 minutes.
  5. Remove (and discard) supernatant.
  6. Incubate the diamond pellet with 50 ul of deionized water containing 2% β-mercaptoethanol.
  7. Roughly sonicate the sample (5 seconds, use water-bath type sonicator).
  8. Incubate in dark for 10 minutes.
  9. Directly add 50 ul of acetonitrile containing 5 ul 4-vinylpyridine into the solution.
  10. Incubate in dark for 10 minutes.
  11. Centrifuge at 12000 rpm for 1 minute (longer time results in tightly packed pellet and may make the following procedure difficult).
  12. Remove (and discard) supernatant.
  13. Wash pellet with 1 ml (can be less) of acetonitrile, spin down (12000 rpm for 1 minute) and discard the supernatant.
  14. Add 50 ul of ammonium bicarbonate containing trypsin (sample/enzyme=50/1 w/w).
  15. Sonicate the sample to disperse precipitated nanodiamonds.
  16. Incubate the sample at 37oC overnight (or at 50oC for 3 hours).
  17. (For LC-MS)Centrifuge at 12000 rpm for 5 minutes, collect and then dry the supernatant under vacuum (for removing residual ammonium bicarbonate). (This portion contains the peptide fragments)Resuspend the dried pellet with 0.1% formic acid prior to LC-MS analysis.
  18. (For MALDI-MS)Dry the protease tube without any workup under vacuum, add matrix solution to the dried residue for direct analysis).

Protein elution(temporary recipe)

Follow steps 1~5 for binding a protein (originating from, say, isolation by gel filtration) from dilute or contaminated working solution. Elute adsorbed proteins with (typically) 30 / 28% NH4OH, protein will disolve and collect the solution. Repeat multiple times if desired, and combine all the supernatant recoverd.

Further information

Please contact Wesley Hsiao atservice@fndbiotech.com