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Methods:TCA precipitation

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Contents

Purpose

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Precipitation of proteins and nucleic acids with trichloroacetic acid is used to measure incorporation of precursors into high molecular weight macromolecules[1], or degradation of macromolecules. TCA precipitation is also used to concentrate and denature proteins for gel electrophoresis or for digestion prior to mass spectrometry [2].

Cold TCA

TCA precipitation is often done with ice-cold solutions of TCA, added to a culture or experimental sample to give a final concentration of 5-10%. The precipitated material is collected on a filter or by centrifugation.

Hot TCA

Hot TCA is used in amino acid labeling experiments to hydrolyze the amino acids on charged tRNAs.

Materials Needed

SOLUTIONS:

100% TCA (w/v); Store at 4C

Acid Acetone (1 part HCl: 39 parts acetone); keep at -20C

Protocols

PROCEDURE:

1. Add 1/10 volume of 100% TCA to sample; mix well

2. Place on ice (or in –20C) for minimum 1 hour

3. Centrifuge at 13,000 rpm (4C) for 5 minutes (length of spin depends on sample volume)

4. Remove supernatant materials & save on ice

5. Rinse pellet with ice cold acid acetone

6. Centrifuge at 13,000 rpm (4C) for 5 minutes (length of spin depends on sample volume)

7. Remove supernatant materials and discard

8. Air dry the pellet

9. Resuspend in buffer of choice

Quantitating incorporation or release of amino acids or nucleotides

Concentrating samples

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References

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  1. Bonifacino, JS (2001) Metabolic labeling with amino acids. Curr Protoc Mol Biol Chapter 10 Unit 10.18 PubMed EcoliWiki page
  2. Kim, SC et al. (2006) A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea. J. Proteome Res. 5 3446-52 PubMed EcoliWiki page