Team:WashU/Protocols/Trypsin

From 2012.igem.org

(Difference between revisions)
(Created page with "{{WashUbackprotocols}} == Trypsin Digestion for LC/MS == #Make, run, stain and destain SDS-PAGE gel as usual. Note: If protein is not being overproduced, purification may be ne...")
 
Line 1: Line 1:
{{WashUbackprotocols}}
{{WashUbackprotocols}}
-
 
+
<div id = "protocolshort">
== Trypsin Digestion for LC/MS ==
== Trypsin Digestion for LC/MS ==
#Make, run, stain and destain SDS-PAGE gel as usual.  Note: If protein is not being overproduced, purification may be necessary to give a clear band.   
#Make, run, stain and destain SDS-PAGE gel as usual.  Note: If protein is not being overproduced, purification may be necessary to give a clear band.   

Latest revision as of 17:24, 17 August 2012


Trypsin Digestion for LC/MS

  1. Make, run, stain and destain SDS-PAGE gel as usual. Note: If protein is not being overproduced, purification may be necessary to give a clear band.
  2. Excise protein band to be investigated using a razor blade. Following excision, mince this band into 0.5mm cubes and place in a low-bind eppendorf tube.
  3. Destain gel pieces by adding 200ul of 200 mM ammonium bicarbonate and 40% acetonitrile and incubating for 30min at 37degrees
  4. Repeat step 3 once more.
  5. Dry gel pieces using a speed-vac for 30min.
  6. Add 20 μl (0.4 μg of trypsin) of rehydrated trypsin to the tube.
  7. Add 50 μl of 40 mM ammonium bicarbonate and 9% acetonitrile to the gel sample and mix.
  8. Incubate mixture overnight.
  9. The peptide pieces are now in the solution is now ready analyze by LC/MS. Simply remove the liquid, carefully avoiding the gel pieces.
Retrieved from "http://2012.igem.org/Team:WashU/Protocols/Trypsin"