Buffer exchange

Crossflow Filters - These are usually membrane-type filters used for ultrafiltration. In the field of biotechnology these types of filters are used in ultrafiltration devices used in concentrating solutions, and performing buffer exchanges. 

Check the column packing quality Buffer exchange before testing... 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

Label a set of tubes according to the standards and samples to be used. Add 250 pi of each standard and sample to the appropriate tubes. If the samples are in a buffer that may significantly change the pH of the reaction buffer, the samples should be buffer-exchanged or dialyzed into 0.1M sodium phosphate, pH 8.0, before running the assay. 

Prepare an amine-modified oligonucleotide according to any of the protocols discussed in Section 2.1 (this chapter). Dissolve or buffer-exchange the oligo into 0.1M sodium borate, 2mM EDTA, pH 8.25, at a concentration of 9 nmol (2.0 A2gonm units) in 15 pi. 

Dissolve or buffer exchange the prey protein into 50mM MOPS, 120mM NaCl, 0.1 mM EDTA, 10mM MgCl2, 10 percent glycerol, pH 8.0. 

As mentioned earlier, the response of each protein will vary. This is especially apparent with colorimetric assays or derivatization methods requiring a chemical reaction. These protein-to-protein reactivity differences mean that a protein assay suitable for one protein may not be suitable for another. Even for a given protein and a specific protein determination method, results may still vary based on limitations of the assay. Methods requiring extensive sample preparation including protein concentration, buffer exchange, and time-sensitive reactions are liable to be less reproducible than direct measurement techniques, which have fewer variable parameters. The application will determine the suitability of the method. 

Controlled metal complexation — While uncontrolled metal complex -ation can be a major source of problems, controlled complexation is a potentially useful selectivity modifier. The key is to evaluate the effects of only one metal at a time, which in turn requires that your sample be stripped of metals in preparation for your experimental treatment. The EDTA-imidazole treatment described above can be used for this purpose. For evaluating the effects of ferric iron or calcium, buffer exchange the treated sample into 0.05 M MES, pH 6, then add the metal salt of choice to a concentration of 5 mM. For other metals, buffer exchange the treated sample into 0.05 M Tris, pH 8, then add the metal salt of choice to 5 mM. 

A wide variety of substituted boronates are available commercially. For phenyl supports, use a phenyl or biphenyl (napthalene) derivative. For nonphenyl supports, evaluate both weakly and strongly hydrophobic alkyl derivatives (straight chain or branched). Raise sample pH to at least 8 and add the boronate derivative of choice to a concentration of at least 1 mM. The sample need not be buffer-exchanged prior to injection. Column buffers should be at least pH 8. 

Recombinant MAb samples (2.5 mg) were buffer exchanged into 800 (il of 0.1 M sodium bicarbonate, pH 8.3, using an NAP-5 column. A measure of 10 pi of 5-TAMRA.SE (1.4 mg/ml) dissolved in DMSO was then added to 190 (il of rMAb solution, and the resultant mixture was incubated at 30°C for 2 h. After incubation, 190 pi of the antibody-dye conjugate was loaded into a second NAP-5 column and collected into 700 pi of 0.1 M sodium bicarbonate, pH 8.3. 

Figure 12.2 Using NMR to monitor the clearance of penultimate buffer components by diafiltration. The traces from top to bottom are the NMR spectrum of the feed to the dialfitration and the spectra of the samples taken after different diavolumes of buffer exchange have been completed. The penultimate buffer components (including HEPES, Tris, and citrate) are clearly removed after 10 diavolumes of buffer exchange.

Most CZE separations are very sensitive to conductivity (e.g., salt concentration) in the run buffer. Therefore, to avoid introducing a high amount of salt from the sample injection, samples should be buffer exchanged with an appropriate low salt buffer prior to analysis. Centrifugal UF/DF devices are ideal for this purpose, as they are typically very reproducible and allow the analyst greater flexibility in controlling the final sample concentration. 

The NAP-25 /PD-10 column contains Sephadex G-25 and is used for a rapid desalting or buffer exchange of nucleic acids, proteins and oligonucleotides. 

Other systems with similar components can also be used, provided they can be operated at flow rates that will be compatible with the column-operating pressures. For some systems, additional column fittings may be required to facilitate connection of the Superose 6 column. If the purpose of the gel-filtration step is to exchange buffers, then the column should be equilibrated and eluted with the buffer that the sample is to be exchanged into. Optimal separation of sample components can be achieved with a sample volume of 200 pL. For desalting or buffer exchange, a sample volume of up to 2 mL can be used. 

In the separation of biomolecules, sample preparation almost always involves the use of one or more pretreatment techniques. With high-performance liquid chromatography (HPLC), no one sample preparation technique can be appHed to all biological samples. Several techiques may be used to prepare the sample for injection. For example, complex samples require some form of preffactionation before analysis, samples that are too dilute for detection require concentration before analysis, samples in an inappropriate or incompatible solvent require buffer exchange before analysis, and samples that contain particulates require filtration before injection into the analytical instrument. 

Penefsky introduced the centrifuged-column procedure (see diagram below) that can be used for small aliquots of enzymes and other proteins (a) to exchange buffers and/or other solutes, (b) to measure ligand binding, and (c) to concentrate protein by as much as 10 times. His two-step protocol for rapid buffer exchange can be summarized as follows ... 

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