We need to conserve our plant samples in nature during the excursion for future analyses of DNA, RNA and proteins. For DNA and RNA it is not a problem, but we have a serious problems with proteins. We want to quantify from tissue plant hormones, but at this moment our data is changing if we compared extracts from fresh (or frozen) tissues with conserved in different solutions. Can anybody have a recommendation? Is it useful e.g. Allprotect tissue reagent from Qiagen?
You can preserve plant sample for PGR analysis by fixing in 80% methanol containing BHT.
Hi Anil,
Thanks for your reply. Is this method published somewhere?
Lenka
If I remember. You should plant material homogenized in liquid nitrogen. Extraction of hormones in 80 % ethanol within 14-16 hours at 4оС in the ratio 1:10 (g:ml)[kudoyarova et al., 1986]. Gomogenat should centrifuged. Supernatant united, transferred to Petri's cups and ethanol evaporated in an air current till the water rest. Alikvota of the rest selected for the quantitative analysis of cytokinins (ELISA method).
The water should rest alkalinized one-percentage NaHCO3 and twice extraction in neutral and main substance by sulfuric ether. Ether extract rejected. The water phase was acidified by 1 M of HCl to рН 3,0 and took hormones in sulfuric ether . Radio extract metylation hormones solution in diazometane on ether. Then ether evaporated on air, the dry rest of hormones dissolved in 80 %-numbers ethanol. After this procedure ABA and IAA is stabilised and can remain long time. But I analized of hormones on ELISA method.
Hi Igor,
Thanks. Liquid nitrogen works very well (and dry ice as well), but there is serious problem with handling during long term excursions...How to replace that??
Lenka
In long term excursion you sould have the metallic thermos or small duar vessel with liquig nitroden or dry ice. In thermos with liqud nitrogen samples can save 4-5 day. One still small cunning. To store samples wrapped in a aluminium foil in liquid nitrogen. It keeps still some time. Only extraction of hormones and its stabilisation it necessary to carry out quickly and according to a technique
Well, it is exactly the procedure which we are using...It seems that another technique does not exist, at least at this moment...
Well, Why do we use 80% of ethanol/ methanol for hormones extraction, instead of absolute. What is the reason behind it ?
For in the water residue (~ 20%) are the main hormones. After the extraction they are should be stabilized.
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