Prince:FASP: Difference between revisions
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==Steps== | ==Steps== | ||
** Slight modifications can/should be made according to the complexity of your sample | ===Protocols=== | ||
* '''Preparing cells/tissue lysate''' | |||
**Lyse cells or homogenized tissue in SDT-lysis buffer using 1:10 sample to buffer ratio at 95°C for 3-5 min | |||
**Incubate the lysed cells for 3 min in boiling water | |||
**Sonicate the lysed cells using Branson SONIFIER 250 for 30 s (Amplitude 40%) | |||
**Incubate the mixture for 3 min in boiling water | |||
**Clarify the crude extract by centrifugation at 16,000 x g at 30°C for 10 min. | |||
* '''Sample processing''' | |||
** ''Slight modifications can/should be made according to the complexity of your sample'' | |||
**Mix the lysate with UA buffer in the ratio of(1 mg protein:1 ml UA buffer) for purified proteins add 200µl of UA-DTT and vortex it | |||
**load samples in 30k filters.(Amicon ultra/ Vivacon 500) | |||
*** ''(Sample loading and speed of centrifugation would depend on the filters used)'' | |||
**concentrate the solution for 15 min at 14000 × g | |||
**Add 100µl of UA and centrifuge the filters for 8 min at 14500 g(twice) | |||
**Add 100µl of UA-IAA, mis it well and incubated at 20° C for 20 min in dark. | |||
**Add 100µl of UA centrifuge the filters for 8 min at 14500 g (twice) | |||
**Add 100µl of ABC and centrifuge the filters for 8 min at 14500 g (twice) | |||
**Add trypsin(proteomic grade)in the ratio (1:40) | |||
** | |||
==Notes== | ==Notes== | ||
| Line 20: | Line 39: | ||
* ABC | * ABC | ||
** 50mM Ammonium Bicarbonate | ** 50mM Ammonium Bicarbonate | ||
Revision as of 12:12, 28 July 2010
FASP Protocol
Template File provides easy calculation of volumes and masses for Solution preparation
Steps
Protocols
- Preparing cells/tissue lysate
- Lyse cells or homogenized tissue in SDT-lysis buffer using 1:10 sample to buffer ratio at 95°C for 3-5 min
- Incubate the lysed cells for 3 min in boiling water
- Sonicate the lysed cells using Branson SONIFIER 250 for 30 s (Amplitude 40%)
- Incubate the mixture for 3 min in boiling water
- Clarify the crude extract by centrifugation at 16,000 x g at 30°C for 10 min.
- Sample processing
- Slight modifications can/should be made according to the complexity of your sample
- Mix the lysate with UA buffer in the ratio of(1 mg protein:1 ml UA buffer) for purified proteins add 200µl of UA-DTT and vortex it
- load samples in 30k filters.(Amicon ultra/ Vivacon 500)
- (Sample loading and speed of centrifugation would depend on the filters used)
- concentrate the solution for 15 min at 14000 × g
- Add 100µl of UA and centrifuge the filters for 8 min at 14500 g(twice)
- Add 100µl of UA-IAA, mis it well and incubated at 20° C for 20 min in dark.
- Add 100µl of UA centrifuge the filters for 8 min at 14500 g (twice)
- Add 100µl of ABC and centrifuge the filters for 8 min at 14500 g (twice)
- Add trypsin(proteomic grade)in the ratio (1:40)
Notes
Buffer Definitions
- UA
- 8M Urea in 0.1M Tris-HCl @ pH 8.5
- UA/DTT
- 0.1M DTT in UA buffer
- UA/IAA
- 50mM IAA in UA buffer
- SDT Lysis Buffer
- 2% w/v SDS, 0.1M DTT in 0.1M Tris-HCl @ pH 7.6
- ABC
- 50mM Ammonium Bicarbonate
Measuring peptide concentration in eluant
- 1mg/ml solution has 1.1 au at 280nm
Concentration of the peptides can be estimated by UV spectrometer assuming that 0.1% solution of vertebrate proteins has at 280 nm an extinction of 1.1 absorbance units.
Reference
FASP paper (from the supplement of Nature Methods: 6(5) 2009)
Trypsin
Why you do not want to have any residual reducing agent (e.g. DTT) in your digestion buffer:
Porcine Trypsin [Green: Backbone, Red: Disulfide bonds]
