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| Samples containing blood (in .5 ml acetonitrile) are extracted for juvenile hormone (JH) by adding 1 ml hexane and 0.5 ml 0.9% NaCl solution. JH ends up mostly (about 98%) in the top hexane phase because JH is more fat soluable (less polar). Viewed: 1084 times. |
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| The tubes are put on ice for 10 min and then centrifuged at 2000 g to give a better separation of the aqueous and hexane phases. Viewed: 979 times. |
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| The hexane phase is easily removed by using a pippet. Viewed: 683 times. |
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| Each sample would have its own pasteur pippet (with a bulb) to avoid cross contamination. Viewed: 1039 times. |
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| We repeat the hexane extraction a 2nd time. The hexane (2 ml) must be dried down to concentrate the JH in the sample. We use a Savant solvent drying system (SS21), which inlcudes a vacuum pump (left), a centrifuge (right), and a cold trap [next photo]. Viewed: 907 times. |
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| The cold trap cools to below 100 degrees C to condense the hexane so it does not ruin the vacuum pump. Viewed: 936 times. |
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| When the vacuum meter shows 300 milli-torr or below, it generally means all hexane is dried off and we can remove the JH tubes. Viewed: 977 times. |
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| The dried tubes (containing JH) are put on ice to minimize JH loss (JH can be oxidized to become JH acid). Viewed: 962 times. |
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| We then add 100 microliter (ul) of methanol to the sample tubes and vortex vigorously to wash the JH from the tube. Viewed: 906 times. |
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| We now take an aliquot (10 ul or 20 ul) out, dry the alcohol, then add 200 ul mixture of JH antibody and radio-labeled JH. JH in the sample will compete with labeled JH to bind to the antibody. Because binding sites are limited, the more JH we have in a sample, the more radiolabeled JH will be displaced from the antibody. Incubation is carried out for 2 hours at room temperature. Viewed: 1021 times. |
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Album: Bee research 
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