Prince:FASP: Difference between revisions

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===Protocols===
===Protocols===
* '''Preparing cells/tissue lysate'''
* '''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
**Lyse cells or homogenized tissue in SDT-lysis buffer using 1:10 sample to buffer ratio
**Incubate the lysed cells for 3 min in boiling water
**Incubate the lysed cells for 10 min at 95°C
**Sonicate the lysed cells using Branson SONIFIER 250 for 30 s (Amplitude 40%)
**Sonicate the lysed cells (Branson SONIFIER 250 for 30 s) (Amplitude 40%)
**Incubate the mixture for 3 min in boiling water
**Incubate the mixture for 10 min at 95°C
**Clarify the crude extract by centrifugation at 16,000 x g at 30°C for 10 min.
**Clarify the crude extract by centrifugation at 16,000 x g at room temperature for 10 min.


* '''Sample processing'''
* '''Sample processing (for complex sample)'''
** ''Slight modifications can/should be made according to the complexity of your sample''
**load 40µl of lysate with 200µl of UA buffer in 30k filters.(Amicon ultra/ Vivacon 500)and vortex it
**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
**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 and centrifuge the filters for 15 min at 14000 × g (twice)
**Add 100µl of UA-IAA, mix it well and incubated at 20° C for 20 min in dark. Spin out the UA-IAA reagent at 14500g for 8 min.
**Add 100µl of UA-IAA, mix it well and incubate at room temperature for 20 min in dark. Spin out the UA-IAA reagent at 14000 × g for 15 min.
**Add 100µl of UA  centrifuge the filters for 8 min at 14500 g (twice)
**Add 100µl of UA  centrifuge the filters for 15 min at 14000 × g (twice)
**Add 100µl of ABC and centrifuge the filters for 8 min at 14500 g (twice)
**Add 100µl of ABC and centrifuge the filters for 20 min at 14000 × g (twice)
**Add trypsin(proteomic grade)to the sample in the ratio (1:40)
**Add trypsin(proteomic grade)to the sample in the ratio (1:40)
*** for incomplete trypsin digestion and better coverage, microwave the sample for 1 min at 20% power in a microwave with an inverter technology.(thrice, make sure the sample cools down before, subsequent microwave)
*** for incomplete trypsin digestion and better coverage, microwave the sample for 1 min at 20% power in a microwave with an inverter technology.(thrice, make sure the sample cools down before, subsequent microwave)
*** for complete digestion carry out an overnight (6-18 hrs) trypsin digestion in addition to the microwave method.
*** for complete digestion carry out an overnight (6-18 hrs) trypsin digestion in addition to the microwave method.


==Notes==
==Notes==

Revision as of 10:37, 22 November 2010

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FASP Protocol

  • Template File provides easy calculation of volumes and masses for Solution preparation
FASP Template

Steps

Protocols

  • Preparing cells/tissue lysate
    • Lyse cells or homogenized tissue in SDT-lysis buffer using 1:10 sample to buffer ratio
    • Incubate the lysed cells for 10 min at 95°C
    • Sonicate the lysed cells (Branson SONIFIER 250 for 30 s) (Amplitude 40%)
    • Incubate the mixture for 10 min at 95°C
    • Clarify the crude extract by centrifugation at 16,000 x g at room temperature for 10 min.
  • Sample processing (for complex sample)
    • load 40µl of lysate with 200µl of UA buffer in 30k filters.(Amicon ultra/ Vivacon 500)and vortex it
    • concentrate the solution for 15 min at 14000 × g
    • Add 100µl of UA and centrifuge the filters for 15 min at 14000 × g (twice)
    • Add 100µl of UA-IAA, mix it well and incubate at room temperature for 20 min in dark. Spin out the UA-IAA reagent at 14000 × g for 15 min.
    • Add 100µl of UA centrifuge the filters for 15 min at 14000 × g (twice)
    • Add 100µl of ABC and centrifuge the filters for 20 min at 14000 × g (twice)
    • Add trypsin(proteomic grade)to the sample in the ratio (1:40)
      • for incomplete trypsin digestion and better coverage, microwave the sample for 1 min at 20% power in a microwave with an inverter technology.(thrice, make sure the sample cools down before, subsequent microwave)
      • for complete digestion carry out an overnight (6-18 hrs) trypsin digestion in addition to the microwave method.

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
    • 4% 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:

Trypsin with disulfide bonds
Trypsin with disulfide bonds

Porcine Trypsin [Green: Backbone, Red: Disulfide bonds]

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