While preparing the suspension from friable callus fragments for desired time period (20-30 days), there is possibility of the dispersion of the cells in the liquid Ms medium, but as per most of the references people first filter the callus suspension -> wash it twice/thrice to remove impurities > dry in oven and powder -> reconstitute in methanol -> Sonicate and use for further analysis.
Is this the accurate procedure ?
Kindly help.
Hi, there are two possibilities for finding the secondary metabolites from suspension cultures cultures; I mean secondary metabolites can be also excreted in the media and or secondary metabolites are not secreted in the media. I agree with the comments above, so I think it will be nice if you can analyze both media and biomass., best regards
We have screened for the extracellular medium http://aobpla.oxfordjournals.org/content/5/plt025.long for our terpenoids and AGP (proteins). We used HPTLC to screen for media-dissolved /secreted terpenoids here.
So, it would not hurt to screen the media, esp. if you know what you are looking for! Many biotechnology products are from media (as the down stream processing becomes cheaper and faster from media processing than breaking cells and getting rid of other contaminants from entire cells!).
So, check both and who knows where your products are!
Thanks,
Hi, there are two possibilities for finding the secondary metabolites from suspension cultures cultures; I mean secondary metabolites can be also excreted in the media and or secondary metabolites are not secreted in the media. I agree with the comments above, so I think it will be nice if you can analyze both media and biomass., best regards
It is possible that people go through these lenghty procedures to clean the sample prior HPLC. This way they can avoid column clogging, etc. Now if you use HPTLC there is no need for extensive sample preparation because you will never use the same HPTLC plate for more then 1 analysis. TLC is very friendly in the sense that it lets you work with very dirty samples.
The procedure will also depend on what you are interested in. Intra- or extracellular compounds?
I would extact the suspension with hexane or another organic solution and use a syringe filter prior to apply something onto a HPLC-Column.
As you know HPLC sample solution must be completely not contain any micro particle to avoid the blockage of the column, so you must done ultrafiltration.
I agree withtge sugestions of the peers. If you are focusing on the phytochemicals found within the cell then their extraction from callus is the obvious procedure. If the desired phytochemicals are extracellular then extraction of them from media will be needed. Analysing both will be of really useful for your research. You have to only optimize the extraction procedure either for callus or media or BOTH. All the best.
- Badges
- Science method