Protein precipitation method

More information on the comparative evaluation of protein precipitation methods may be obtained from Lei and coworkers.163 An interesting comparison of protein precipitation (PPT) and solid phase extraction (SPE) methods was presented in a technical library publication from Millipore164 that describes use of its Multi-SPE-MPC extraction plate and Multiscreen deep well Solvinert filter plate for SPE and PPT, respectively (Figure 1.45). A Biohit Proline multichannel pipette was used to add 400 /iL of acetonitrile to each well of the filter plate and then, using the pipette s double aspiration program, 100 /iL of spiked serum was aspirated and 100 /iL of acetonitrile from the filter plate was aspirated to initiate protein precipitation in the pipette tip. The mixture was deposited back in the filter plate and shaken vigorously for 2 min. 

Jiang, L., He, L. and Fountoulakis, M. (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. Journal of Chromatography A 1 023, 31 7-320. 

Direct solvent extraction before or after acid or enzymatic digestion has almost superseded die classic protein precipitation methods. Due to die more sensitive detection methods of gas chromatography and mass spectrometry, much smaller amounts of tissue can be processed. Consequently, any emulsion problems that arise are more easily resolved than in the past when several hundred grams of liver and large volumes of solvent were required. 

C. J. Kitchen, A. Q. Wang, D. G. Musson, A. Y. Yang, and A. L. Eisher, A semiau-tomated 96-well protein precipitation method for the determination of mon-telukast in human plasma using high performance liquid chromatography/ fluorescence detection, J. Pharm. Biomed. Anal. 31 (2003), 647-654. 

Each of these approaches is practical for performing high throughput protein-precipitation methods. The determining factors for selection of the collection-plate format over the filtration plate, or vice versa, include the extent of available hardware and automation accommodating the microplate format, total cost of materials, and thus the cost per sample, number of physical manipulations, and the degree of transfer loss deemed acceptable. 

Hagerman, A. E. Butler L. G. Protein precipitation method for the quantitative determination of tannins. J. Agric. Food Chem. 1978,26,809-812. 

The concentration of protein in all lysates should be determined using a protein assay (e.g., Ettan 2-D Quant Kit) that is compatible with typical 2-DE denaturing reagents present in the lysate solution. Protein concentration of 5-10 mg/ml in the lysate is ideal. However, samples containing 1 mg/ml protein have been successfully labeled using the protocol below. Use a protein precipitation method (e.g., Ettan 2-D Clean-up kit) to concentrate the sample(s) if necessary. 

As mentioned before, the amount of soluble tannin that causes astringency in persimmon fruits is usually estimated visually by the tannin print method and can be measured quantitatively by the Folin-Denis method. There is also a protein precipitation method for the measurement of soluble tannins (Hagerman and Butler 1978). In that method, the soluble tannin content is assayed by the addition of the sample to a standard solution of protein and the isolation of insoluble tannin-protein complexes. The complexes are dissolved in alkaline solution, to which ferric chloride is added. The absorbance of the solution at 510 nm is measured. 

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