|
|
| 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: 1702 times. |
|
|
|
| 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: 1549 times. |
|
|
|
| The hexane phase is easily removed by using a pippet. Viewed: 1211 times. |
|
|
|
| Each sample would have its own pasteur pippet (with a bulb) to avoid cross contamination. Viewed: 1600 times. |
|
|
|
| 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: 1671 times. |
|
|
|
| The cold trap cools to below 100 degrees C to condense the hexane so it does not ruin the vacuum pump. Viewed: 1487 times. |
|
|
|
| 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: 1541 times. |
|
|
|
| The dried tubes (containing JH) are put on ice to minimize JH loss (JH can be oxidized to become JH acid). Viewed: 1508 times. |
|
|
|
| We then add 100 microliter (ul) of methanol to the sample tubes and vortex vigorously to wash the JH from the tube. Viewed: 1452 times. |
|
|
|
| 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: 1586 times. |
|
Album: Bee research 
1 
2 
3 
