Prince:FASP: Difference between revisions
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{{PrinceLab}} | {{PrinceLab}} | ||
=FASP Protocol= | |||
Template File provides easy calculation of volumes and masses for Solution preparation | |||
[[Image:Example.jpg]] | |||
==Steps== | |||
** Slight modifications can/should be made according to the complexity of your sample | |||
==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 | |||
===Protocols=== | |||
* Preparing cells/tissue lysate | |||
**Lyse cells or tissues 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. | |||
===Measuring peptide concentration in eluant=== | ===Measuring peptide concentration in eluant=== | ||
* 1mg/ml solution has 1.1 au at 280nm | |||
<blockquote>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.</blockquote> | |||
===Reference=== | |||
FASP paper (from the supplement of [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1322.html Nature Methods: 6(5) 2009]) | |||
===Trypsin=== | ===Trypsin=== | ||
Why you do not want to have any residual reducing agent (e.g. DTT) in your digestion buffer: | |||
Why you do not want to have reducing agent (e.g. DTT) in your digestion buffer: | |||
[[Image:Trypsin porcine with disulfide.png|center|Trypsin with disulfide bonds|300px]] | [[Image:Trypsin porcine with disulfide.png|center|Trypsin with disulfide bonds|300px]] | ||
Porcine Trypsin [Green: Backbone, Red: Disulfide bonds] | Porcine Trypsin [Green: Backbone, Red: Disulfide bonds] | ||
Revision as of 11:17, 28 July 2010
FASP Protocol
Template File provides easy calculation of volumes and masses for Solution preparation
Steps
- Slight modifications can/should be made according to the complexity of your sample
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
Protocols
- Preparing cells/tissue lysate
- Lyse cells or tissues 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.
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]
