Prince:Yeast FASP: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
| Line 12: | Line 12: | ||
===Sample Processing=== | ===Sample Processing=== | ||
* 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 | ||
* Wash sample with 10 ml of UA buffer and centrifuge for 40 minutes at 3000g '''(twice)''' | * 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 | * 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 10 ml UA buffer and centrifuge 3000g 40 minutes '''(twice)''' | * Wash with 10 ml UA buffer and centrifuge 3000g 40 minutes '''(twice)''' | ||
* Wash with 10 ml ABC buffer and centrifuge at 3000g for at least an hour and half '''(twice)''' | * Wash with 10 ml ABC buffer and centrifuge at 3000g for at least an hour and half '''(twice)''' | ||
* Protein on small portion of sample assay if desired | * Protein 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) | ||
* Pulse sample filters to 1000g | * Pulse sample filters to 1000g | ||
* Incubate sample filters at 37 °C in a rotating incubator for 18 hours | * 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 | * Clean the collection portion filter tube very well with distilled water. After washing rinse with mass spec water | ||
* Centrifuge at 3000g for 30 minutes | * Centrifuge at 3000g for 30 minutes | ||
* 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 | ||
* Acidify with .1% Formic Acid unless fractionating with SCX or SAX | * Acidify with .1% Formic Acid unless fractionating with SCX or SAX | ||
Revision as of 17:59, 12 January 2012
- Template File provides easy calculation of volumes and masses for Solution preparation
Cell Lysis
- Incubate yeast cells at 96°C for 10 minutes
- Add 10% 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
- Centrifuge samples at 16000g to remove cell lysates
Sample Processing
- 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 10 ml UA buffer and centrifuge 3000g 40 minutes (twice)
- Wash with 10 ml ABC buffer and centrifuge at 3000g for at least an hour and half (twice)
- Protein 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
