Dialysis buffer exchange

Instead of dialysis, small molecules, such as salts or organochemi-cal substances, can be separated quickly from macromolecules by gel filtration. Buffer exchange is also possible this way. The disadvantage is a dilution of the sample. 

If necessary, remove surplus reagents and/or make buffer exchange by gel filtration on Sephadex G-25 or dialysis. Concentrate the eluate and the dialysate, respectively, to about 1 ml. 

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. 

In industry, dialysis is not widely used because it is slow and labor-intensive. Here, desalting and/or buffer exchange is usually performed by diafiltration or molecular exclusion chromatography, which are discussed later. 

Osmotic driven (dialysis) methods Against solutions Against water absorbing materials Few pLs to few mLs Small-scale Mechanically gentle Rapid Simultaneous buffer exchange Concentrations achieved may not translate to manufacturing scale Have to buffer exchange later Yes... 

Ammonium sulfate can easily be removed from the precipitated antibody preparations by dialysis of the solution against large volumes of the desired buffer. Gel filtration or size-exclusion chromatography is another method used for salt removal and buffer exchange. 

Gel filtration is a rapid and efficient alternative to dialysis for desalting and buffer exchange. It is used, for example, in the preparation of protein samples prior to freeze drying or fractionating by ion exchange chromatography. Applications include ... 

For long-term storage of the piurified scFv preparation it is advisable to buffer exchange the sample to remove the imidazole which can lead to sample instability. This can be achieved either by dialysis (gainst PBS) or by use of a Napp 5 Sephadex C25 column (Pharm a Biotech) following the manufacturer s instructions. 

It is not normally possible to capture antibodies direct from culture supernatant because the concentration of competing salt is too high. A preliminary buffer exchange step is necessary. A wide range of buffers might be suitable and buffer exchange is usually achieved by dialysis or diafiltration. Some antibodies precipitate under these conditions which can reduce the overall yield. Several options might be explored in such a case (1) ... 

In the application of FRET to detect and quantify protein-target interactions, the protein and the target are each labeled with either a donor or an acceptor prior to being brought into contact. The probes can be conjugated to the macromolecule via primary amine or sulfhydryl or other appropriate chemistries. In general, the unattached probes should be carefully removed by dialysis or other means of buffer exchange. This requirement can be eased when a chelated lanthanide ion is used as the donor. 

A major nonhemodialysis application of countercurrent dialysis (CCD) is the buffer exchange of proteins in the biopharmaceutical industry. In multistep biopharma-ceutical production processes, the buffer solution constituents often have to be exchanged for protein purification steps. The original buffer solution, for example, may contain, say, 0.5 M ammonium sulfate and 0.05 M sodium phosphate (plus protein and other constituents) the CCD... 

To implement buffer exchange, coutercurrent dialysis has been explored to eliminate 0.05 M sodium pbospbate from a 1 mg/ml solution of bovine serum albumin (BSA) and 25 mM Tris buffer at a pH of 7 (via NaOH). Tbe dialyzing buffer composition is 25 mM Tris and tbe pH is 7 (via HCl). The dialysate flow rate is 100 ml/min. Two membrane modules are in series, each having a surface area of 0.2 m. The value of (Ci/o/Cj/i) for sodium phosphate at a feed flow rate of 10 cm /min was found to be 246. Determine the value of the overall mass-transfer coefficient Kg. (Ans. 1.5 x 10 cm/min.)... 

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