Edman Sequencing Sample Preparation and Submission

Samples for sequencing may be submitted as PVDF bands blotted from SDS-PAGE gels and then stained, or as a solution of >80% pure protein. Electroblotting is preferred because it allows selection of the band of interest and removes contaminants that may be present in solution. Solution samples may contain buffer salts but not detergent. Amount of protein recommended for sequencing is at least 10 pmoles, or a band easliy visible by Coomassie staining.

Electroblotting Proteins onto PVDF for N-terminal Sequencing

Proteins may be transferred to polyvinyldifluoride (PVDF) membranes from SDS gels for N-terminal sequencing.  Recommended PVDF membranes are those from Millipore (Immobilon Psq), Applied Biosystems (ProBlott), and BioRad.  Nitrocellulose may NOT be used for sequencing.

Procedures for electroblotting:

1. Prepare CAPS 3-[cyclohexylamino]-1-propanesulfonic acid buffer.
   
   a. 10X CAPS (100mM, pH 11): Dissolve 22.13 g of CAPS in 900 mL of D.I. water.  Titrate with 2N NaOH (approx. 20 mL) to pH 11, and add D.I. water to a final volume of 1 liter.  Store at 4° C.
   b. Electroblotting buffer (1X stock buffer in 10% MeOH): Prepare 2 liters of buffer by mixing 200mL of 10X CAPS buffer with 200mL methanol and 1600mL D.I. water.
   
2. Wet PVDF with methanol for a few seconds, and place the membrane (the size of the gel) in a dish containing blotting buffer.
3. Remove the gel from the electrophoresis cell and soak it in electroblotting buffer for 5 minutes.
4. Assemble the sandwich, and electroblot at constant voltage of 50 volts (170 mA – 100 mA) at room temperature for 30 minutes.
5. Remove the PVDF from transblotting sandwich and rinse with D.I. water prior to staining.

Procedure for Coomassie Blue Staining:

1. After rinsing with water, saturate the PVDF with 100% methanol for a few seconds.
2. Stain the PVDF with 0.1% Comassie Blue R-250 in 40% MeOH/1% acetic acid.  Protein bands should appear within one minute.
3. Remove the PVDF from staining solution and destain with 50% MeOH.  CAUTION: Staining for more than one minute may result in a longer destaining time.
4. Rinse extensively with D.I water.  Air dry.
5. The entire blot may be stored at -20° C or bands of interest may be excised with a clean razor blade and placed in Eppendorf tubes.
   
   

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