Separation buffer electrophoresis

Figure 3.12 Metabolic profiling by capillary electrophoresis, (a) Comparative carbohydrate profiles of M. truncatula tissue obtained using 4-aminobenzonitrile derivatization, capillary electrophoresis with a 150 mM borate buffer, pH = 9, and on-column UV detection at 214 nm. (b) Anion profile from M. truncatula using capillary electrophoresis and indirect UV detection. The separation buffer was 5 mM K2C1O4, 1% Waters OFM-Anion BT, pH 8.0.

Separation is performed using free-zone electrophoresis, where the capillary is filled with a separating buffer at a defined pH and molarity. This buffer is also called a BGE. During separation, the polarity is set to cathodic or anodic mode, also called normal and reverse mode, depending on the charge of the molecule cation or anion. For anions, the capillary is usually dynamically coated with an electroosmotic flow (EOF) modifier to reverse the EOF and separate the analytes in the co-electroosmotic mode. 

In the first dimension, 20 [tl plasma is separated by electrophoresis at 4°C in a 0.75% agarose gel using a 1 2 16 dilution of a barbital buffer. Bromophenol blue is added to a standard sample to visualize albumin in the native gel. The electrophoresis is stopped when the albumin/bromophenol blue marker has migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins are then transferred to a 4-20% polyacrylamide gradient gel. Separation in the second dimension is performed at 40 mA for... 

The acid-base properties, and hence ionic character, of peptides and proteins also can be used to achieve separations. Ion-exchange chromatography, similar to that described for amino acids (Section 25-4C), is an important separation method. Another method based on acid-base character and molecular size depends on differential rates of migration of the ionized forms of a protein in an electric field (electrophoresis). Proteins, like amino acids, have isoelectric points, which are the pH values at which the molecules have no net charge. At all other pH values there will be some degree of net ionic charge. Because different proteins have different ionic properties, they frequently can be separated by electrophoresis in buffered solutions. Another method, which is used for the separation and purification of enzymes, is affinity chromatography, which was described briefly in Section 9-2B. 

Multiple column switching has been made possible by the construction of a cyclic CE microstructure in which four channels that are 20 mm in length are arranged to form a square, as illustrated in Figure 8.20.62 Two channels at each corner connect the loop to external reservoirs filled with separation buffer. Platinum electrodes are inserted into the reservoirs for the application of high voltages. One side of the device has a volume-defined injection scheme incorporated into it. The overall technique is called synchronized cyclic capillary electrophoresis.63... 

One of the first steps in modifying the performance of capillary electrophoresis was the deactivation of silica groups of the capillary column by physically coating the capillary wall with methylcellulose (58,59), as well as via silane derivatization (10,44,60). Presently, many other changes have been carried out either to the capillary surface or addition of chemical agents to the separation buffer (see Table II), including manipulation of... 

TABLE II. Improvements on the Capillary Electrophoresis Separation of Proteins, Peptides and Amino Acids by Deactivation of the Silica Surface or by Addition of Chemical Agents to the Separation Buffer... 

Capillary isotachophoresis (CITP) — An electrophoretic separation technique (-> electrophoresis) in a discontinuous -> buffer system, in which the analytes migrate according to their -> electrophoretic mobilities, forming a chain of adjacent zones moving with equal velocity between two solutions, i.e., leading and terminating electrolyte, which bracket the mobility range of the analytes. Ref [i] Riekkola ML, Jonsson jA, Smith RM (2004) Pure Appl Chem 76 443... 

Finally, if the product of the enzymatic reaction is known, the activity of the enzyme can be assayed on the basis of quantitating the product off line. Thus, for instance, glutathione peroxidase activity can be measured by quantitating the oxidized and reduced forms of glutathione by capillary electrophoresis, as demonstrated by Pascual et al. (1992). The electrophoretic separation buffer used was 100 mM tetraborate (pH 8.2), containing 100 m M SDS. 

Fig. 10. Hybridization of GAPDH s (a) rabbit (R) and lobster (L) muscle, pig (P) and lobster (L) muscle (b) rabbit (R) and yeast (Y), pig (P) and yeast (Y). The hybrid bands were separated by electrophoresis on cellulose acetate in 50 mM phosphate buffer, pH 7.0, and revealed in (a) by protein staining and in (b) by activity staining (cf 103).

Figure 4-20. Schematic principles of various electrophoretic separation methods. In zonal electrophoresis (left) particles move according to their electrophoretic mobilities towards the oppositely charged electrode separation into discrete bands depends on mobility differences. In isotachophoresis (centre), the electrode and separation buffers are different. Particles in the sample form tight bands or zones, ordered according to electrophoretic mobility,...

Gramicidin A, however, is attacked by JV -bromoacetamide (NBA) and N-bromosuccinimide (NBS) (Gross and Witkop, unpublished observation). In 50 % aqueous ethyl alcohol at room temperature 5 % of the peptide bonds (20% of the tryptophyl peptide bonds) are cleaved with NBS. Methyl alcohol must be avoided because it opens the spirodioxindole lactone from oxidized tryptophan to the ester even at room temperature. The cleavage mixture separates on electrophoresis (pH = 2.5, sodium-formate buffer) into four ninhydrin-positive components of which the fastest migrating one was identified as ethanolamine. Dinitrophenylation showed leucine and alanine to be additional NHs-terminals of the released fragments. 

The iS-peptide of subtilisin-modified ribonuelease (Richards and Vitha-yathil, 1959) was cleaved with cyanogen bromide and the fragments separated by electrophoresis in three different buffer systems. The peptide... 

Unlike HPLC, sample preparation in CE requires careful thinking and strategy to obtain a good analysis. There is a relationship between the sample matrix and the separation buffer. Based on how the sample is prepared and how the separation buffer is selected, sample matrix effects can be both favorable and detrimental to the analysis. Matrix effects are different in capillary zone electrophoresis (CZE) from those observed in micellar electrokinetic capillary chromatography (MEKC). Understanding sample matrix effects is the first step in sample preparation in order to obtain a good separation by CE [1]. 

For gel purification, 20CM-00 pmol of 32P-labeled RNA crude mixture is mixed as a tracer with 40,000 pmol of unlabeled crude substrate. Measure the radioactivity of a small aliquot of the mixture in a scintillation counter to calculate the specific activity of the RNA. The dsRNA and ssRNA in the crude mix are separated by electrophoresis in a preparative 8% polyacrylamide gel. After visualization by autoradiography, the region of the acrylamide gel containing the dsRNA is excised with a razor blade and the RNA is extracted from the gel. After gel purification, the dsRNA substrate is suspended in buffer I, and the radioactivity of an aliquot is measured on the scintillation counter. From the specific activity of the dsRNA, the amount of gel-extracted substrate can be determined. This is called the cold substrate because the specific activity is lower than that of the hot substrate, which is freshly labeled with 33P for use in the assay. Since a small amount of the purified dsRNA is labeled with 32P, aliquots of gel-purified cold substrate should be stored in an acrylic (3-radiation storage container until they the radiation has decayed with time. 

Because CE uses online optical detection, artifacts can result in the form of system peaks. These often originate from the sample or the interfaces between the sample and the separation buffer because any species that absorbs at the detection wavelength wfll generate a response. This differs from protein slab gel electrophoresis where detection specificity is governed by a protein specific stain. It is not uncommon, for example, for buffer species present in the sample but not in the separation buffer to generate system peaks. However, clinical serum protein electrophoresis provides one example where artifacts are elimmated by CE. 

Electrophoretic separation on agarose gels or cellulose acetate membranes is the procedure most commonly used to demonstrate LD isoenzymes." After the isoenzymes have been separated by electrophoresis, a reaction mixture is layered over the separation medium. The mixture (typically D, L-lactate> 500mmol/L, and NAD, 13mmol/L, often dissolved in a suitable pH 8.0 buffer) is applied as a liquid or in a gel. The NADH generated over the LD zones is detected either by its fluorescence, when excited by long-wave ultraviolet light (365 nm), or by its reduction of a tetrazolium salt to form a colored formazan. 

FIGURE 4 Indirect laser-induced fluorescence detection of 19 amino acids by microchip electrophoresis. Separation buffer 1.0 mM sodium carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH at pH 10.3. Separation condition /eff 5.5 cm, 15 s injection at 417 V/cm (reversed polarity), 183 V/cm separation voltage, sample amino acid concentrations of 0.4 mM in 1.0 mM sodium carbonate and 0.2 mM CTAOH. Reprinted with permission from [66]. Copyright 2000, The American Chemical Society. 

Fig. 11 Capillary zone electrophoresis of recombinant erythropoietin glycoforms under denaturing conditions. The separation buffer consisted of 0.010 M tricine, 0.01 M NaCl, 0.01 M sodium acetate, 7 M urea, and 2.5 mM putrescine, pH 5.5. (For details of experimental conditions, see Refs. 410 and 411.)...

For vertical systems the gel column is inserted between two separate buffer reservoirs the upper reservoir contains the cathode, and the lower one contains the anode. Gel electrophoresis is usually carried out at basic pH, where most biological polymers are anionic and hence they move down toward the anode. The sample to be analyzed is layered on top of the gel and voltage is applied to the system. 

Ribosomal proteins are soluble in concentrated salt solutions. Most of them can be dissolved without damage by buffers containing 2 M LiCl and can then be separated by electrophoresis or ion exchange chromatography and gel filtration (molecular sieving). Although many of them are quite insoluble and are often unstable, all ribosomal proteins of E. coli (Table... 

Most CE work so far has been done using the capillary zone electrophoresis (CZE) mode, where analytes are separated on the basis of differences in electrophoretic mobility, which is related to charge density. The separation is carried out in a capillary filled with a continuous background electrolyte (buffer). Micellar electrokinetic capillary chromatography (MEKC or MECC) is one other CE method based on differences in the interaction of the analytes with micelles present in the separation buffer, which can easily separate both charged and neutral solutes with either hydrophobic or hydrophilic properties. An alternative to MEKC is capillary... 

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