Hello,
In most modern protocols for metabolomics/lipidomics proteins are precipitated using solvents such as methanol, acetonitrile or isopropanol.
After vortexing/mixing the next step is often to put the samples in the freezer for varying times. I am interested in learning more about this step since there seems to be no consensus regarding how it should be done. Some researchers do 15 minutes others an hour, some even more. I do not expect this to have a huge impact on the results but would be interested to see an actual comparison regarding time in freezer (and different properties of different solvents at lower temperatures).
I am also interested in what is happening to the samples chemically at the lower temperature. I understand the need to keep the samples cold to minimize the extent of enzymatic conversion of compounds but it is sometimes stated that the cold solvent and the keeping of samples in the freezer enhance precipitation. I have not seen a reference for this and would appreciate to read a thorough discussion.
Looking forward to any responses!
Take care, stay healthy and keep doing science!
Hi henrick,
Mentiontation in freezer: This is simply due to decrese in solubility product o f spairingly soluble salt with decrease in temperature. After additin of organic solvent like metanol, acetone etc. most of the electrolytic components (like bicarbonate, magnesium chloride and other salts) behave as spairingly soluble salts. At freezer i.e below -20oC temperature solubility product of such type of electrolyte decreases in a certain extent and easily seperated out with precipitated protein. Now you can get maximum amount of backgroun free supernatent. Here the incubation time depends on your targated analyte of interest.
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1023293/pdf/jclinpath00008-0001.pdf
https://www.sciencedirect.com/topics/chemistry/protein-precipitation
https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1907.sp001234
Thanks
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