Hello all!
Does anyone know a protocol that i can use to determine whether a medium contains any fatty acids or sterols?
I don't really need to identify or measure the levels of these "contaminants". All i want is to check if they are present or not!
Onengiyeofori Ibama just a simple HBSS medium with glucose, magnesium and amino acids added in it. I dont mind ”destroying” the mediUm. I just wanna see whether there are any lipids in it.
Given that this is a liquid growth medium and it is not expected to contain remarkable amount of fat, I would suggest to extract the total fat (i.e. crude lipid) following Bligh and Dyer method.
To a sample containing 1 ml HBSS medium in a clean glass tube with screw top lid, add 3.75 ml of a mixture chloroform:methanol (1:2) and vortex for approximately 10 min. Now add 1.25 ml chloroform then mix for 1 min, then add 1.25 ml water and mix for another minute before centrifugation for 15 minutes at approximately 4000 rpm. The sample in the tube will form clearly separate phases in the following order from the top: water, protein and organic solvent. Discard the upper aqueous phase and collect the lower organic solvent phase (containing fat) through the thin protein layer with a Pasteur pipette. Weigh the organic solvent part in a new clean glass tube, then evaporate the sample by a rotary evaporator if you have access to one, or by using a nitrogen blower in a fume hood. After evaporation, the lipid extract will remain and you will be able to know the weight by subtracting the difference before and after evaporating the organic solvent. Watch some tutorials online for more details.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology