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'''Method #1 -- TCA extraction''' To obtain intracellular protein-free lysate: Homogenize tissue and lyse cells in 5% final concentr..."

2012-12-18T21:49:54Z

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'''Metabolite extraction'''
 <HR> 
'''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.

 <HR> 
'''Method #2 -- ProteinPurification kits'''

Kits are available from BioVision, National Diagnostics, CalBiochem, and SigmaAldrich to name a few.

Here is an example:[[Image: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)

[[Image:Biovision.png]]

 

 <HR> 
'''Method #3 -- Methanol extraction'''

Sample Preparation - Revision history

Wikiroot: Created page with "'''Metabolite extraction''' '''Method #1 -- TCA extraction''' To obtain intracellular protein-free lysate: Homogenize tissue and lyse cells in 5% final concentr..."

Wikiroot: Created page with "'''Metabolite extraction'''

'''Method #1 -- TCA extraction''' To obtain intracellular protein-free lysate: Homogenize tissue and lyse cells in 5% final concentr..."