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The procedure described below is an alternative protocol for extraction of oxytocin from aqueous solutions.
1. Add two volumes of ice cold acetone to the sample. Vortex.
2. Centrifuge at 12,000 x g for 20 minutes.
3. Remove the supernatant and transfer to a fresh tube.
4. Add five volumes of ice cold petroleum ether. Vortex.
5. Centrifuge at 10,000 x g for 10 minutes.
6. Discard top ether layer. Carefully transfer remaining aqueous layer to a glass tube and dry down under nitrogen or argon gas.
7. Reconstitute sample with Assay Buffer.
2. Centrifuge at 12,000 x g for 20 minutes.
3. Remove the supernatant and transfer to a fresh tube.
4. Add five volumes of ice cold petroleum ether. Vortex.
5. Centrifuge at 10,000 x g for 10 minutes.
6. Discard top ether layer. Carefully transfer remaining aqueous layer to a glass tube and dry down under nitrogen or argon gas.
7. Reconstitute sample with Assay Buffer.
Please note that recovery of peptides from the extraction processes cay vary. It is important to optimize the process to obtain optimum recoveries. Extraction efficiencies can be determined by a number of methods, including the use of radioactive peptide, or by spiking into paired samples and determining the recovery of this known amount of added oxytocin.