Protein salting-out

J. Laitinen, J. Samarut and E. Holtta, A nontoxic and versatile protein salting out method for isolation of DNA, Biotechniques, 17 (1994) 316-322. 

C. J. Coen, J. M. Prausnitz and H. W. Blanch, Protein salting-out phase equilibria in two-protein systems, Biotechnol. Bioeng. 1997, 53, 567-574. 

Solution behavior of polymers and proteins, salting out, precipitation, extraction, chromatography, resin swelling, phase splitting etc. General relevance for DSP... 

S ts can be used to precipitate proteins by salting out effects. The effectiveness of various salts is determined by the Hofmeister series, with anions being effective in the order citrate > PO4" > SO4" > CH3COO > Cl > NO3 , and cations according to NH4 > > Na ... 

Extracellular Enzyme Activities. The protein, carrier of the polygalacturonase and pectinmethylesterase activities, was salted out from the H.annuus 1805 culture medium by adding ammonium sulphate to 70 % saturation. The precipitate was separated by centrifugation (30 min, 4500 xg), diluted with 0.1 M phosphate buffer (pH 7.0) and dialyzed for 12 hours against distilled water. After dilution to the required volume with the same buffer, both enzyme activities under study were determined in the solution. 

Proteins that are polyampholytes, upon addition of electrolyte, first undergo some salting in up to a certain ionic strength, since electrostatic interactions between ions are shielded by the additional simple ions of both sign. Adding more salt will then cause salting out as the added ions compete for water that would otherwise solvate the protein. 

Salting-out salts (Na2S04, NaCl, MgS04) Surface tension increase Weak binding Stabilize globular proteins and precipitants of native and denatured proteins... 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

Protein precipitation is used routinely in bioanalytical laboratories in the pharmaceutical industry. Plasma is mixed with an excess (3 to 5 times) of organic solvent (typically acetonitrile or methanol) or an acid such as formic acid. The proteins precipitate out of solution, the sample is centrifuged, and the supernatant is analyzed. While this method is relatively fast and simple, the extract contains salts and lipids that can interfere with subsequent analyses. Often, a second technique such as SPE is used for further cleanup. Table 2.4 exhibits various samples that... 

Many cytosolic proteins are water soluble and their solubility is a function of the ionic strength and pH of the solution. The commonly used salt for this purpose is Ammonium Sulphate, due to its high solubility even at lower temperatures. Proteins in aqueous solutions are heavily hydrated, and with the addition of salt, the water molecules become more attracted to the salt than to the protein due to the higher charge. This competition for hydration is usually more favorable towards the salt, which leads to interaction between the proteins, resulting in aggregation and finally precipitation. The precipitate can then be collected by centrifugation and the protein pellet is re-dissolved in a low salt buffer. Since different proteins have distinct characteristics, it is often the case that they precipitate (or salt out ) at a particular concentration of salt. 

Effect of salt type and concentration The ionic strength of the aqueous solution in eontaet with a reverse micelle phase affects protein partitioning in a number of ways [18,23]. The first is through modification of electrostatic interactions between the protein surface and the surfaetant head groups by modifieation of the eleetrieal double layers adjacent to both the eharged inner mieelle wall and the protein surface. The second effect is to salt out the protein from the mieelle phase because of the inereased propensity of the ionie speeies to migrate to the micelle water pool, reduee the size of the reverse mieelles, and thus displace the protein. 

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