Prince:Yeast FASP

• Template File provides easy calculation of volumes and masses for Solution preparation

FASP Template

Cell Lysis

• Place eppendorf containing yeast cells in boiling water.
• Add 10% boiling SDS to yeast cells 1:1 (v/v) for a 5% total SDS concentration. Incubate at 96°C for 10 minutes
• Add DTT to .1 M concentration
• Sonify samples at 50% amplitude for 1 minute 8 times with a 30 second delay between sonications in 1.5 ml or 2 ml eppendorfs(you may have to divide samples to prevent overflow during sonication)
• Centrifuge samples at 16000g to remove cell lysates

Sample Processing (for samples > 1mg protein)

• Dilute supernatant with UA buffer to 10 ml. Load into Vivaspin 15R 10k filters. Centrifuge at 3000g for 50 minutes
• Wash sample with 5 ml of UA buffer and centrifuge for 40 minutes at 3000g (twice)
• Add 2 ml UA-IAA, mix well, incubate at room temperature in the dark for 20 minutes. Centrifuge at 3000g for 30 minutes
• Wash with 5 ml UA buffer and centrifuge 3000g 40 minutes (twice)
• Wash with 5 ml ABC buffer and centrifuge at 3000g for at least an hour (can take as long as 2.5 hours) (twice)
• Protein assay on small portion of sample assay if desired
• Add trypsin (proteomic grade) to the sample in the ratio (1:40) (make sure at least 1 ml of ABC is in the filter during trypsin addition)
• Pulse sample filters to 1000g
• Incubate sample filters at 37 °C in a rotating incubator for 18 hours
• Clean the collection portion filter tube very well with distilled water. After washing rinse with mass spec water
• Centrifuge at 3000g for 30 minutes
• Rinse with 1 ml ABC. Centrifuge 30 minutes at 3000g
• The flow through contains the peptides
• Speedvac to desired volume
• Acidify with .1% Formic Acid immediately prior to mass spec analysis