Prince:In Gel FASP

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Method

Gel Excision and Shredding

Excision is essentially the standard protocol

Water bottle (MilliQ water), Lexan sheet used as a cutting surface, and box of razor blades, all shown in a laminar flow hood to reduce potential contaminants to each excised band.

* Excise band using clean razors and surface (image at right suggests necessary materials/workspace requirements)

* Place excised band into a P200 tip, add 100 μL (MilliQ water) and centrifuge (~5000g) into a tube using something like Centrifuge Adapters

* [Optionally] Load shredded gel into P10 tip and repeat.

Pre-FASP

* Load sample of shredded gel onto a FASP appropriate filter (As indicated at FASP Protocol Page)

* Destain the protein band by addition of 200 μL of 1:1 Acetonitrile : UA Buffer for 25 minutes

* Centrifuge at 14000 g to remove destain solution

• Continue with FASP by adding UA/DTT and subsequent steps, noting that greater centrifugation times might be required to fully wash each sample

as developed from July 28th through August 3rd, 2010 by *Ryan M Taylor 14:36, 3 August 2010 (EDT):

Cautions

• Watch the fluid levels post-centrifugation to ensure the elution was sufficient prior to moving to the next step
• Do not excise too large of a band to ensure centrifugation steps do not take much longer (2-3 fold longer)

Qualification

Excision of three bands of 2.0 mg/mL BSA standards (21 μL: Heavy, 7 μL: Light, and 0.1 μL: Tiny) run on a 10% Acrylamide Gel resulted in 93%, 85%, and 83% sequence coverage by Mascot search of data acquired on a LTQ Orbitrap XL in the Prince Lab over the dates indicated.