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Team:Evry/Experimentals Parameters
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| - | <p>To determine half-life time of auxin in tissues, tissue needs to be soaked in a great volume of MMR medium with auxin, during 20 minutes, to uptake the hormone.<br/> | + | <p><br/>To determine half-life time of auxin in tissues, tissue needs to be soaked in a great volume of MMR medium with auxin, during 20 minutes, to uptake the hormone.<br/> |
<i>Estimation of auxin concentration in the tissue is possible, due to the strong difference between the volume of tissue and the bath. </i><br/> | <i>Estimation of auxin concentration in the tissue is possible, due to the strong difference between the volume of tissue and the bath. </i><br/> | ||
Revision as of 02:26, 27 September 2012
Protocols to determinate the following experimentals parameters
Auxin distribution in tadpole
To determine where the auxin gather in tadpole, you have to inject the auxin directly in it's heart. In this way it will be distributed with the bloodstream. The next stop is tadpole quenching and detection of the auxin distribution by fluorescent anti-auxin antibody, which are comercially available.
Determination of auxin diffusion from tadpole's skin to the medium
To determine the coefficient of auxin diffusion from the skin to the medium (MMR), tadpole's skin is soaked in MMR+auxin medium for 20minutes. After that tissue is transferred to MMR solution without auxine, and increase of auxin concentration in MMR is observed. It is thus possible to plot a curve (auxin quantity in fonction of time ).
Determination of auxin diffusion from blood to muscle
To determine the coefficient of auxin diffusion from blood to muscle, inject auxin directly into the tadpole's heart. 5min of recuperation time for tadpole in MMR is required. Afterwards euthanize the tadpole, collect muscle and identify the auxin concentration in samples.
Determination of auxin half life in tissues (muscle and skin )
To determine half-life time of auxin in tissues, tissue needs to be soaked in a great volume of MMR medium with auxin, during 20 minutes, to uptake the hormone.
Estimation of auxin concentration in the tissue is possible, due to the strong difference between the volume of tissue and the bath.
Then place the tissus loaded in a small volume of MMR with the same auxin concentration.
The volume of solution has to be equal to the volume tissue.
Here we know how much auxin was present in the bath of small volume.
Apply the wait time ( the first is 5 minutes, it will increase after ), determine the auxin present in the tissue and the environment, to finaly subtract it from the initial quantity of auxin in tissue plus medium
Then simply subtract the degradation rate with tissue from MMR only, to know the amount of auxin degraded by tissue volume in 5 minutes
Repeat this experiment with at least 10 tadpoles, and by increasing the incubation time in the bath small volume of 5 minutes each time.
Finally just assign the value of auxin degradation over time to determine the parameters
How to determine the Auxin Half Life in the Blood
About the half-life time of auxin in blood, you just have to get a volume of blood, and mix that with an auxin known quantity. Wait 5 minutes and determine the concentration of auxin.
Subtract the initial quantity of auxin added from the quantity mesurate in blood after 5 minutes to get the auxin degraded in the blood during the 5 minutes.
Restart the experiment ten time, increasing the wait time by 5 minutes to each time to trace the curve of the auxin degraded in blood fonction of time.
