How to Use Sanger Reagents

Weigh out 3 miligrams of peptide on a scale. Place it in a centrifuge tube. Add approximately 0.3 ml of water, 0.3 ml of Sanger's Reagent and 0.075 ml of sodium bicarbonate.

Allow the centrifuge tube to incubate at room temperature for one hour. During the incubation, shake the tube gently to prevent the mixture from separating. Check the pH of the solution every 15 minutes with pH paper. The pH should be kept above 9. If it begins to drop, add a small amount of sodium bicarbonate (approximately one drop with the Pasteur pipette).

After the incubation, add 1.5 ml of water and an equal amount of ether. Allow the solution to separate into layers. You may have to centrifuge the mixture to separate it. Remove the ether layer (the top, yellow layer) with a pipette and discard it.

Adjust the pH down to 1.0 by slowly adding hydrochloric acid. Add a little bit of acid at a time, stir gently, then check the pH with pH paper. Altogether, it should require about 0.15 ml of acid. If the pH goes below 1.0, add a small amount (no more than a drop) of sodium bicarbonate.

Add 3 ml of ether. Allow the mixture to separate. Now the top layer (the yellow, ether layer) contains your DNP-compound. Using a pipette, carefully extract this layer and transfer it to a clean test tube. Place the test tube aside and allow the liquid to evaporate, leaving behind a dry, yellow solid.

Wash your solid with acetone. Add 0.3 mililiters of acetone, stir gently and extract the liquid with a pipette. Add 0.75 mililiters of acid to the test tube. Your peptide is now tagged and ready to be hydrolized and analyzed with the chromatography method of your choice.