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On-Blot, In Situ Digest

   
This procedure was adapted from the following article:
Wong, S. C., Grimley, C., Padua, A., Bourell, J. H., and Henzel, W. J. (1993) Peptide Mapping of 2-D Gel Proteins by Capillary HPLC. Tech. In Prot. Chem. IV, 371-378

Reagents:

  1. Methanol
  2. Acetonitrile
  3. Water
  4. DTT, Clealands reagent
  5. Tris-Base
  6. HCl
  7. Promega modified trypsin
  8. Iodoacetic acid (IAA) (see tips on this reagent, step 4 on this link)
  9. PVP,  polyvinylpyrolodone 

Solutions and Stocks:

  1. 1 M DTT (in water, keep this stock frozen at -20 蚓)
  2. 0.5 M Tris-Base / 6M guanidine-HCl / 5 mM EDTA / 10% acetonitrile / 7 mM DTT /  pH 8.5 with HCL (note: pH with HCl before adding the acetonitrile, starts out around pH 10. Add DTT fresh to a portion of this solution right before reduction)
  3. 200 mM IAA in 0.5 M NaOH make fresh each time
  4. 10% Acetonitrile
  5. 0.25% PVP  in 0.1% acetic acid
  6. 100 mM Tris-Base, pH 8.0, 10 % acetonitrile

Procedure:

  1. Cut out the band or spot as narrowly as possible, avoid touching the blot, wear gloves.
  2. Wet excised blot with 1-3 無 of methanol in a small tube.  (the membrane is very hydrophobic and must be wetted with methanol.  Do not let the membrane dry out during the procedure.)
  3. Reduction.  Reduce with 100 無 of Solution 2 at 45 蚓 for 60 min.
  4. Cool and add 10 無 of Solution 3, let react for 20 min. in the dark.
  5. Wash the blot 3X with 500 無 Solution 4.
  6. To block the blot incubate in 200 無 of Solution 5 for 20 minutes on a room temp. shaker.
  7. Wash the blot 3X with 500 無 Solution 4.
  8. Transfer blot to new tube.
  9. Wash the blot 3X with 500 無 Solution 4. (this extensive wash and transfer is necessary to remove excess PVP 360, which will give UV and mass spectral interference if not removed.)
  10. Digest with 0.2 痢 Promega modified trypsin in 100 無 of Solution 6 for 24 hours at 37 蚓.
  11. Concentrate on a SpeedVac to 20 痞, inject 5 無 onto a capillary column for LC/MS analysis.

 

 

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Figure 1
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Figure 2
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Figure 3
 
 
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Figure 4

 

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Figure 5

 

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Last updated:  Friday, April 29, 2005 08:17:36 AM