Practical Protein Chemistry - A. Darbre 1989

Micro-scale amino acid sequence analysis using a gas-phase peptide-protein sequencer
Polypeptide purification for microanalysis
Instrumentation and Methodology

To unfold the polypeptide chain, the sample was heated for 5 min at 80 °C in 0.08 M Tris-buffer (pH 6.8) containing 2% SDS, 75 mM dithiothreitol (Calbiochem), 10% (v/v) glycerol, and 0.05% bromothymol blue. After cooling to room Temperature, sodium thioglycolate was added to the sample to a final concentration of 5 mmol/L, followed by SDS-PAGE [10]. Upon completion of Electrophoresis, the gels were stained for 15 min in a solution containing 20% isopropanol, 10% acetic acid, and 0.5% Coomassie Blue, and then destained at 4 °C in a solution containing 25% methanol and 10% acetic acid. The gel slice containing the Coomassie-stained protein band was excised with a razor blade, cut into small cubes, and left to swell in distilled Water for 1 h to remove acetic acid and methanol. Excess liquid was drained from the gel, and the gel particles were allowed to swell for 2–16 h at room temperature in 0.5–1.0 mL of a 0.2 M ammonium bicarbonate solution containing 1% SDS and 0.1% dithiothreitol. This Treatment neutralizes residual acidity and resaturates the protein with SDS.

The design of the electrophoretic eluter-concentrator includes two plexiglass chambers for sample loading and concentration, each closed at the bottom with a dialysis membrane; the chambers are connected to each other by an electrolytic bridge. The device is similar in design to the commercial Isco model, with the exception that the smaller (concentration) chamber is widened at the bottom to match the diameter of the larger (loading) chamber. This design feature facilitates protein concentration through the membrane of the smaller chamber, but at the same time worsens the conditions for SDS concentration, since the surface areas of the membranes through which ions migrate into and out of the eluter are equal.

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FIG. 17.6. Yields of PTH-Amino Acid Derivatives during N-terminal sequence analysis of 5 nmol, 500 pmol, and 70 pmol of angiotensin II (sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). Data Processing as described in the legend to Fig. 17.5.

The gel particles and the equilibration solution containing 1% SDS are transferred into the loading chamber of the eluter. The device is filled with 0.05 M ammonium bicarbonate containing 0.1% SDS and 0.5 mM sodium thioglycolate. The electrode vessels are filled with the same solution. Electrophoresis is carried out for 16 h at 50 V with buffer recirculation; then the electrode buffer solution is replaced with 10 mM ammonium bicarbonate containing 0.02% SDS. Electrophoresis is continued for another 20 h at 80 V. Under these conditions, the eluted protein is quantitatively concentrated into a solution volume of <100 µL. The dense zone of Coomassie Blue and protein is removed from the dialysis membrane using a microsandwich/Hamilton microsyringe. The sample is then applied to The surface of the Sequencer Glass fiber filter.