Prince:Yeast FASP

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Strong Cation Exchange)
Line 26: Line 26:
* The flow through contains the peptides
* The flow through contains the peptides
* Acidify with .1% Formic Acid unless fractionating with SCX or SAX
* 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)
 

Revision as of 09:13, 13 September 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
Personal tools