This is for pre-processing, before the derivatization step.
I used trichloroacetic acid 50%, but after spinning down I seem to lose some significant amount of media sample and the result does not seem reliable.
Hello Anusha,
I see that TCA 50% is to remove/ precpitate out proteins in media and some salts/coloring agents etc. Not sure from your question as to how that lads of significant amount of media loss? Please share the protocol that you used for better answers.
Also, worth giving a read to the following excellent works where:
(1) cell type: adherent/ non-adherent
(2) quenching methods
(3) washing steps
(4) other optimizations
(5) choice of platform: your GCMS vs LCMS
have a been reviewed/ discussed nicely:
Removing the bottlenecks of cell culture metabolomics: fast normalization procedure, correlation of metabolites to cell number, and impact of the cell harvesting method: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025493/
Experimental design and reporting standards for metabolomics studies of mammalian cell lines.: https://link.springer.com/article/10.1007%2Fs00018-017-2582-1
Cell culture metabolomics: applications and future directions.: https://www.ncbi.nlm.nih.gov/pubmed/20601091
Effects of culture media on metabolic profiling of the human gastric cancer cell line SGC7901 : http://pubs.rsc.org/-/content/articlelanding/2015/mb/c5mb00019j/unauth#!divAbstract
Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231.: https://www.ncbi.nlm.nih.gov/pubmed/28497155
Thanks and best wishes,
Biswa
1:1 ratio of ice cold 100% methanol (final concentration is 50% methanol). Counter extraction with chloroform if you wish, then tryptophan/kyn will be in the polar phase.
We do this frequently.
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