Prince:Yeast FASP

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(Strong Cation Exchange)
Current revision (09:16, 13 September 2012) (view source)
(Sample Processing (for samples > 1mg protein))
 
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===Sample Processing (for samples > 1mg protein)===
===Sample Processing (for samples > 1mg protein)===
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* Dilute supernatant with UA buffer to 15ml.  Load into Vivaspin 15R 10k filters.  Centrifuge at 3000g for 50 minutes
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* Dilute supernatant with UA buffer to 10 ml.  Load into Vivaspin 15R 10k filters.  Centrifuge at 3000g for 50 minutes
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* Wash sample with 10 ml of UA buffer and centrifuge for 40 minutes at 3000g '''(twice)'''
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* 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
* 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 UA buffer and centrifuge 3000g 40 minutes '''(twice)'''
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* Wash with 5 ml ABC buffer and centrifuge at 3000g for at least an hour '''(twice)'''
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* 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
* 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)
* 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)
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* Rinse with 1 ml ABC.  Centrifuge 30 minutes at 3000g
* Rinse with 1 ml ABC.  Centrifuge 30 minutes at 3000g
* The flow through contains the peptides
* The flow through contains the peptides
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* Acidify with .1% Formic Acid unless fractionating with SCX or SAX
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* Speedvac to desired volume
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* Acidify with .1% Formic Acid immediately prior to mass spec analysis

Current revision

  • 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
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