Sample Prep/ Concentrating the Sample
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These are some notes from my good friend Sachiko!
Her work on bioreporters/biosensors: http://wiki.biodesign.cc/wiki/Main_Page
POCIS
http://www.est-lab.com/pocis.php
C18 silica gel
- http://www.materialharvest.com/welcome/silica_products/functional_and_reverse.html
- https://tools.thermofisher.com/content/sfs/gallery/high/89870-001-C18-Columns.jpg
- http://www.mn-net.com/Portals/4/images/Redakteure_Chroma/HPLC/C18-Pyramid-3D.gif
suppliers
- http://www.silicycle.com/products/functionalized-silica-gels/chromatographic-phases/reversed-phases
- https://www.sorbtech.com/chromatography/adsorbents/silica-gel/
DIY versions...
Separation Methods for Waste and Environmental Applications By Jack Watson (not enough detail here: packets of silica gel (desiccant, in nori-packets, to keep moisture out) - heated to “sufficiently high” temperatures will lose its -OH groups and “become hydrophobic")
Check out::: HYDROPHOBIC fumed micro silica - THIS IS NOT dessicant
"Micro silica is hydrophilic so it is treated with siloxane or silicon oil to make it markedly hydrophobic. These treated powders will float a bead-head fly because their chemical bonds actively repel water." aka Floatants :: seems like fishing stores and archery shops (to keep the feathers of the arrows from getting wet) carry this stuff
my suggestion to try:
- wash everything you will use with acetone (or nail polish remover) -POLYSTYRENE will melt, polypropylene (eppendorfs, pipette tips, etc. won’t) glass containers may be good, but the seal may melt, etc.
- make a column with this stuff Floatants - http://www.chemistryviews.org/details/education/2040151/Tips_and_Tricks_for_the_Lab_Column_Packing.html
- pour acetone - (nail polish remover) and see if it degrades/dissolves - this is washing the Floatants with the solvent that would eventually use to wash off your estrogen
- see if after acetone, it maintains hydrophobicity (repels water= i.e. pour some bottled water on it, and see if the water rolls off) then it’s good to go - this is to check if the “hydrophobic” material on the silica gel is also washed off with acetone…you don’t really want extra hydrophobic stuff coming off - commercial C18 silica gels have hydrophobic alkanes chemically bound to the gels, so they don’t come off
- take 1 part acetone-washed Floatants gel, 9 parts of your river water, shake it up for some time (ask Urs to make you a shake-it-baby - well, something that keeps the Floatants suspended) - like some hours, or over night
- then centrifuge down the Floatants (or let it settle)
- pour out the river water (keep it in case!)
- add a small amount of acetone and shake it vigorously
- take the acetone out (pipette) and put it in an acetone-pre-washed eppendorf
- repeat 8, and pool into 9
- your negative control would be the same total volume of your acetone (to make sure your nail polish remover doesn’t have some steroids in it)
- air dry the acetone in the eppendorfs - now you should have a concentrated hydrophobic compounds from river water
- you can dissolve this in tiny amounts of DMSO and try your YES assay
Tipo de post
BlogPublication date
02/06/2016