ElectroBlot Protocol for Edman Sequencing

Mass spectrometry and tandem MS/MS

Protein Transfer Protocol for Edman Sequencing

ElectroBlot Protein Transfer protocol for Edman Sequencing

HMC Macromolecular Core Facility

Anne Stanley aes7@psu.edu

Transfer protocol suggested:

We recommend the VWR Fluorotrans 0.2 micron membrane by Pall. If you have an old lots of BioRad PVDF (pre-1997) you may still use that, but the newer BioRad PVDF membranes do not appear to have the same quality. The preferred size of the blot piece to put on the sequencer is anything smaller than 2mm square. Using the least amount of PVDF possible is advantageous, since the more PVDF in the reaction chamber the higher the background will be. Submitting two 2 pieces of PVDF will not be better since the ratio of PVDF/sample will stay the same.

The secret of a good gel.... Let the gel polymerize over night. Prerun at 3mA constant current for 2 hours prior to loading the sample. (UNPOLYMERIZED ACRYLAMIDE IN YOUR GEL CAN BLOCK THE N-TERMINI OF YOUR PROTEINS, MAKING EDMAN SEQUENCINGIMPOSSIBLE, see similar warnings for example at http://www.pitt.edu/~biohome/Dept/Frame/proteinlab.htm or http://www.protein.iastate.edu/nsequence494.html)

If your protein is not too basic:

Use CAPS 3-[cyclohexylamino]-1-propane sulfonic acid as the transfer buffer.

10X CAPS (100mM, PH11)

1-Dissolve 22.13 g of CAPS in 900ml of DI water. Titrate with 2N NaOH to pH 11 and add DI water to 1000ml. Electroblotting buffer (1X stock buffer in 10% MeOH): Prepare 2 liters of buffer by mixing 200ml of 10X Caps buffer with 200ml of MeOH and 1600ml of DI water.

2-Wet PVDF with MeOH for a few seconds and place membrane in a dish containing blotting buffer.

3-Remove gel for the electrophoresis cell and soak in electroblotting buffer for 5 minutes.

4- Assemble the sandwich and electroblot at either constant voltage of 50Volts at room temp. for 30 minutes or constant current of 500 mA for the same amount of time.

5- remove PVDF and rinse with DI water.

Coomassie staining

1. Saturate PVDF with MeOH for a few seconds

2. Stain PVDF with 0.1% coomassie Blue R250 in 40% MeOH/1% acetic acid for 30 seconds.

3. Remove PVDF and destain with 50% MeOH.

4. Rinse with water.

If your protein is very basic:

Use a regular Tris-Glycine buffer and after staining wash the blot extensively with plain H2O.

Example Tris-Glycine buffer - 25 mM Tris, 190 mM glycine, 0.1% SDS, 20% methanol, pH 8.5

To make 1000 ml, dissolve 14.4 g of glycine, 3.0 g of Trizma base, and 1.0 g of SDS in 800 ml of deionized water. Add 200 ml of methanol, stir and degas for 20 min. This buffer should be prepared freshly.

Note that for basic proteins it is possible to need to use blotting membrane on BOTH sides of the gel. Proteins that are more basic than the pH of the transfer buffer will be captured on the cathode side membrane. Those less basic will be captured on the anode side. Increasing the pH of the Tris-Glycine transfer buffer to pH 9.2 will make all proteins below pI ~9.2 transfer toward the anode electrode, etc.