Desalting protein solution

Desalting or buffer exchanges are often required between purification steps. At the laboratory scale, the protein solution is placed in a tube of a semipermeable polymer membrane immersed in the desired buffer. The membrane pore size determines the minimum molar mass of the compounds that are retained. Small molecules with a molar mass below the membrane cut-off will flow freely across the membrane until the osmotic pressure equilibrium is reached. Complete buffer exchange requires several changes of the dialysis liquid. The process should be carried out at a temperature around 4°C, to avoid loss of activity. 

The protein solution is concentrated and desalted against KP 6.2 in Centricon-10 filter units (Amicon, Danvers, MA) as directed by the manufacturer. 

Desalting (removal of low-molecular weight compounds from protein solutions)... 

On-line microdialysis desalting of protein solutions [29] or in-between EC and ESI-MS [30-31],... 

Proteins (IgG, enzymes) can be biotinylated with activated biotin (e.g., photobiotin, NHS-biotin). Particularly useful are the water-soluble sulfo-NHS esters synthesized by Pierce Co. which eliminates the necessity of potentially harmful organic solvents. The reaction occurs via a nucleophilic attack of an amine (usually lysine e-amino groups) towards the ester, yielding a stable amide bond and the release of NHS as a by-product. This reaction is notably effective at higher pH (primary amine unprotonated). Protein should be dissolved at 10 mg/ml and the ester at 0.1 M (fresh solution) in 0.1 M sodium carbonate buffer (pH 8.5-9.0). Ester solution is then added to the protein solution (60 xl/ml for IgG 5 p,l/ml for POase 20 fjLl/ml for BGase). It is not recommended for APase (inactivation of the enzyme BSA can be biotinylated at 20 il/ml and then co-po-lymerized with APase to obtain highly active large complexes). The reaction mixture is incubated for at least 4 h and then desalted on Sephadex G-25. Commercially available complexes are expensive. 

Nl. Nitschmann, H., Kistler, F., Renfer, H., HSssig, A., and Joss, A., A heat stable human plasma protein solution obtained by desalting. Vox Sanguinis 1, 183 (1956). 

Wilson, D.J., Konermann, L., Ultrarapid desalting of protein solutions for electrospray mass spectrometry in a microchannel flow device. Ana/. Chem., 11, 6887-6894, 2005. 

Separate species in the liquid phase whose molar masses differ by factor of 1000 via bulk CS (example, desalt a solution of proteins, we can separate species in the liquid phase whose molar masses differ by at least 20% via SEC. Use SEC for gas phase separation if the relative volatility (based on vapor pressure of key components) a p is < 1.1. See Distillation Section 4.2. 

The first industrial application of SEC for protein solutions were for desalting dairy products (66). Large columns (2500 liters) were used to separate proteins in whey or skim milk from low-molecular-weight sugars and salts. SEC is also used in the deethanolization of human serum albumin (HSA) (67) produced by the Cohn cold ethanol procedure. The purification of insulin was the first successful industrial application of SEC for protein fractionation (68), followed by the fractionation of HSA proteins (69). 

Desalt by gel filtration on a Pharmacia NAP-5 column into 50 mM ammonium bicarbonate buffer, pH 8.5. Load the protein solution in 500-/zl volume using the ammonium bicarbonate to dilute the reduction/... 

Fig. 2. BA-binding activity of the soluble CCKBP. Isolated chloroplasts were suspended in 2 M NaCl with 0.05 M Tris-HCL buffer (pH 7.6). The suspension was incubated in an ice bath for 15 min followed by centrifugation at 23 000 Xg for 10 min. The supernatant was desalted with Sephadex G-25. The proteins eluted by Tris buffer were subjected to (NHJ..SO4 fractionation. The pellets were suspended in Tris buffer and then dialyzed against Tris buffer at 4°C for 4 h. Usually a 1 ml sample (ca. 500 jug protein) was added to an Eppendorf tube containing 0.12 /iCi p H]-BA. After 30 min at 4°C the mixtures were dialyzed for 8 h (at 4°C) against Tris buffer containing 10 mM NaCl and 0.5% charcoal powder. The radioactivity in each sample was counted with 0.2 ml dialyzed protein solution, plus 7 ml of liquid scintillation solution...

Precipitation has some disadvantages as a purification technique for proteins. For dilute protein solutions, yields may be poor [1,9]. Also, the sample may be recovered in a state incompatible with a following purification step. For example, a protein fraction recovered after ammonium sulfate precipitation will require desalting prior to ion-exchange chromatography. 

Removal of salt from a protein solution may be necessary prior to chromatography. For example, injection of high concentrations of salt would result in irreproducibility in retention of early eluting peaks on an ion-exchange column. Concentrated salts may also be incompatible with some organic mobile phases in reversed-phase chromatography. Common desalting techniques are listed in Table 8. 

The most extensive work on intelligent polymers has been carried out on stimuli-responsive hydrogels, particularly those based on pH-sensitive and thermally-sensitive monomers. Their most important applications are related to desalting and/or dewatering of protein solutions, delivery of drugs, on-off immobilised enzyme reaetors, microrobotics and artificial muscle. 

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