you are here » Technological services

« back to previous page

Mass Spectrometry Unit

Samples submission: Guidelines for Proteomics sample submission/prep

INTACT PROTEIN MOLECULAR WEIGHT

Please make certain you have a minimum of 20 uL of protein, at a concentration of at least 5 nanogram/uL. The buffer should contain no urea, no detergents. Below there are a few protocols on how to clean/concentrate a protein sample for mass spec submission.

Protein precipitation protocols

Acetone Precipitation

Most universal. We recommend you use this before any of the others
  • Add to 1 volume of protein solution 4 volumes of cold acetone. Mix and keep 10min
    -70°C and at -20°C for a minnimum of 4 hr or overnight.
  • Spin 15min 4°C in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).
  • Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.

Chloroform/Methanol Precipitation

Useful method for Removal of salt and detergents

The protocol is adjusted for a protein sample volume of 100 Ál. If you have a small amount of protein in a large volume to begin with, it may be better to concentrate the sample before precipitation.
  • Add 400 µl MeOH to 100 Ál protein sample. Vortex and spin down.
  • Add 100 µl CHCl3. Vortex and spin down.
  • Add 300 µl H2O. Mix and spin 1 min at 14000 g. There will now be two phases in the tube with chloroform in the lower phase.
  • Remove the upper phase (aqueous), but take care not to disturb the interphase where your protein is.
  • Add 300 µl MeOH. Vortex and spin for 2 minutes at 14000 g. Discard the supernatant and dry protein pellet in speed-vac.
  • Pellet can be frozen at -20°C or resuspended in your preferred buffer. Reference: Wessel, D. and Flugge, U. I. Anal. Biochem. (1984) 138, 141-143

Acidified Acetone/Methanol

Useful method to remove acetone and methanol soluble interferences like SDS
  • Prepare acidified acetone: 120ml acetone + 10µl HCl (1mM final concentration).
  • Prepare precipitation reagent: Mix equal volumes of acidified acetone and methanol and keep at -20°C.
  • To one volume of protein solution add 4 volumes of cold precipitation reagent. Mix and keep at -20°C minimum 4hr or overnight.
  • Spin 15min 4°C in microfuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).
  • Dry samples under vaccum (speed-vac) or dry air to eliminate any acetone or methanol residue (smell tubes).

Ethanol Precipitation

Useful method to concentrate proteins and removal of Guanidine Hydrochloride before PAGE-SDS
  • Add to 1 volume of protein solution 9 volumes of cold Ethanol 100%. Mix and keep at least 60min.at -20°C. (Suggestion: leave ON).
  • Spin 15min 4°C in microcentrifuge at maximum speed (15000g). Carefully discharge supernatant and retain the pellet: dry tube by inversion on tissue paper (pellet may be difficult to see).
  • Wash pellet with 90% cold ethanol (keep at -20°C). Vortex and repellet samples 5min at full speed.
  • Dry samples under vacuum (speed vac) or dry air to eliminate any ethanol residue (smell tubes). For PAGE-SDS, resuspend samples in a minimal volume of sample buffer.
update: Oct 2010
more info about this unit:
 
last update: February 14, 2012 . Copyright © IFOM & IEO . Campus IFOM-IEO . Via Adamello 16 . 20139 Milan Italy
webmasteratifom-ieo-campus.it - optimized: 1024x768, supported browsers: IE6+ . Safari 4+ . Firefox 2+ . Opera 8+ . Netscape 7+