Prince:Yeast FASP

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===Cell Lysis===
===Cell Lysis===
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* Incubate yeast cells at 96°C for 10 minutes
+
* Place eppendorf containing yeast cells in boiling water.
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* Add 10% SDS to yeast cells 1:1 (v/v) for a 5% total SDS concentration.  Incubate at 96°C for 10 minutes
+
* 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
* 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)
* 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
* Centrifuge samples at 16000g to remove cell lysates
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===Sample Processing===
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===Sample Processing (for samples > 1mg protein)===
* Dilute supernatant with UA buffer to 15ml.  Load into Vivaspin 15R 10k filters.  Centrifuge at 3000g for 50 minutes
* Dilute supernatant with UA buffer to 15ml.  Load into Vivaspin 15R 10k filters.  Centrifuge at 3000g for 50 minutes

Revision as of 15:18, 6 June 2012

  • 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 15ml. Load into Vivaspin 15R 10k filters. Centrifuge at 3000g for 50 minutes
  • Wash sample with 10 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 (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
  • Acidify with .1% Formic Acid unless fractionating with SCX or SAX

Strong Cation Exchange

  • Wash SCX column with 1 ml methanol
  • Wash SCX column with 1 ml .1 M HCl
  • Load SCX column with slightly acidified sample (collect flow through, collect following washes)
  • Wash SCX column with 1 ml .1 M HCl
  • Wash SCX column with 1 ml .1 M HCl in MeOH
  • Elute SCX column with .5 ml 5% NH4OH in MeOH (Twice) combine elutions
  • Add 400 ul of H2O to fractions containing MeOH
  • Speed vac fractions to 400 ul
  • Peptide Assay
  • Concentrate sample to about 2 ug of peptides/ul using speed vac (you may combine low abundance fractions)
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