Sample Preparation
Metabolite extraction
Method #1 -- TCA extraction
To obtain intracellular protein-free lysate:
Homogenize tissue and lyse cells in 5% final concentration trichloroacetic acid (TCA). Incubate on ice about 10 minutes.
Centrifuge for 15 minutes at 14,000 x g at 4°C.
Transfer supernatant to fresh centrifuge tube. pH neutralize with NaOH.
Flash freeze protein-free lysate with liquid nitrogen and store frozen until analysis.
To obtain extracellular protein-free lysate:
Remove cells from medium by centrifugation.
Transfer supernatant to fresh centrifuge tube, add 1/10 volume 50% TCA (to achieve 5% final concentration).
Follow protocol as outlined above.
Note: Frozen protein-free lysate can be lyophilized if desired. The dry lysates can then be redissolved in a smaller volume (to concentrate the sample) or in D2O to gain optimal water suppression in your NMR experiment.
Method #2 -- ProteinPurification kits
Kits are available from BioVision, National Diagnostics, CalBiochem, and SigmaAldrich to name a few.
Here is an example:File:NDkit.pdf
Here is a web site: http://www.biovision.com/deproteinizing-sample-preparation-kit-3826.html
And another: http://vgn.uvm.edu/outreach/documents/539180.pdf
See how this works - spectrum1 is serum - spectrum 2 is hahn spin echo - spectrum 3 is Biovision kit (not on same sample)
Method #3 -- Methanol extraction
