Prince:Yeast FASP: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 4: | Line 4: | ||
===Cell Lysis=== | ===Cell Lysis=== | ||
* | * Place eppendorf containing yeast cells in boiling water. | ||
* 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 | ||
===Sample Processing=== | ===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 12:18, 6 June 2012
- Template File provides easy calculation of volumes and masses for Solution preparation
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)
