Prince:In Gel FASP

=Method=

Gel Excision and Shredding

 * Excision is essentially the standard protocol [[Image:Gel_excision.JPG|thumb| Water bottle, 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 (as the image at right demonstrates)
 * * Place excised band into a P1000 tip and centrifuge into a tube using something like Centrifuge Adapters
 * * Load shredded gel into P200 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= =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.
 * 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)