Prince:FASP

=FASP Protocol=
 * Template File provides easy calculation of volumes and masses for Solution preparation
 * FASP Template

Cell/Tissue Samples:

 * Preparing cells/tissue lysate
 * Lyse cells or homogenized tissue in SDT-lysis buffer using @ a 1:10 sample:buffer ratio
 * Incubate the lysed cells for 10 min at 95°C
 * Sonicate the lysed cells (Branson SONIFIER 250 for 30 s) (Amplitude 40%) until no longer viscous (DNA broken)
 * 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)       times are for 30K filters
 * load up to 40 µl (30μL is best) of supernatant 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)
 * mix the required amount of trypsin in 50µl of ABC and add to the filter, vortex it well and pulse the filters to 2000 × g
 * Incubate the samples for 4 h-18 h at 37 °C
 * Transfer the filter units to new collection tubes and centrifuge at 14000 × g
 * wash the filter with 40µl of ABC and centrifuge at 14000 × g
 * Acidify the solution to 1% formic acid

Purified Protein Samples:

 * Sample processing (for purified proteins)           times are for 30K filters
 * load ~20 µg (have done up to 100) of purified protein in a 1:4 sample:UA buffer ratio in 30k filters and centrifuge for 15 min at 14000 × g
 * Add 100µl of UA-DTT and mix the solution thoroughly centrifuge for 10 min at 14000 × g
 * Add 100µl of UA and centrifuge for 10 min at 14000 × g
 * 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 15 min at 14000 × g (twice)
 * Add trypsin, (0.8 µg proteomic grade) to the sample in the ratio (1:40)
 * mix the required amount of trypsin in 40 µl of ABC and add to the filter, vortex it well and pulse the filters to 2000 × g
 * microwave (inverter based) the sample at 20% power for 1 min and let it cool to room temperature repeat the microwave process again.
 * Transfer the filter units to new collection tubes and centrifuge for 15 min at 14000 × g
 * wash the filter with 40µl of ABC and centrifuge for 15 min at 14000 × g
 * Acidify the solution to 1% formic acid

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

Ordering Information

 * Trypsin
 * Sequencing Grade Trypsin from Promega (C/N #V5113)
 * Filters
 * Sartorius stedim Vivacon 500 Line               Vivacon Product Information
 * 100K
 * 50K
 * 30K
 * 10K   requires additional 30 minutes/spin
 * 2K    we haven't tried yet

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]

=In Gel FASP Protocol= Also, try our new and improved In-gel FASP method!