Prince:In Gel FASP
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Gel Excision and Shredding
- Excision is essentially the standard protocol
- * 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.
- * 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):
- 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)
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.