Cellulose acetate strips

Figure 50-2. Technique of cellulose acetate zone electrophoresis. A A small amount of serum or other fluid is applied to a cellulose acetate strip. B Electrophoresis of sample in electrolyte buffer is performed.

Electrophoresis on cellulose acetate strips (Sepraphore III, Gelman Instrument, Ann Arbor, MI) was done in the conventional manner [12] in order to obtain a comparative electrophoretic mobility of non-adsorbed albumin. For this purpose, BSA-BSA (2.5 w/v) was deposited on the cellulose acetate paper twice in volumes of 10 ul each. Electrophoresis was attain performed in the Gelman Chamber with Pt electrodes at 20°C (see Table 3) After completion, the strips were stained with Ponceau S protein stain (Gelman Instruments) and washed with 5 acetic acid. The stained cellulose acid strips were subsequently cut into 3 mm wide pieces which were monitored for protein content y-count-ing. 

Electrophoresis on cellulose acetate strips has also been used for the rapid resolution of whey proteins (Bell and Stone 1979). Samples of a 10 1 concentrate of whey are applied to cellulose acetate strips which have been saturated with Tris-barbiturate buffer, pH 8.6, ionic strength 0.097, and the electrophoresis is performed at 225 V for 1 hr. This procedure separates not only the major whey proteins but also their genetic variants. 

It seems desirable to encourage other means of determining proteins than by photometry of dyed conjugates, and in this respect direct ultraviolet absorptiometry is apparently an important method (Tl). For this purpose ethanol-treated cellulose powder and cellulose acetate strips are suitable, while buffers satisfactory both for good separation and for ultraviolet reading will be needed. 

Wet a cellulose acetate strip (Schleicher and Schiill, West Germany) 3 cm x 55 cm with pH 3.5 urea buffer (this is made by mixing 47.5 ml 7M urea, 2.5 ml glacial acetic acid, 0.5 ml 0.5M-EDTA). 

Most practical applications of electrophoresis in biochemistry employ some form of zonal electrophoresis, in which the aqueous ionic solution is carried in a solid support and samples are applied as spots or bands of material. Paper electrophoresis, cellulose acetate strip and cellulose nitrate strip, and gel electrophoresis are all examples of zonal... 

For the grafting reactions, solutions were prepared in glass sample tubes (7.5 X 1.6 cm) solvent, sulfuric acid (to 0.2M), and monomer were added to give a total volume of 8 mL and preweighed cellulose acetate strips then were immersed completely in the solutions. Irradiations were performed in air in the spent Fuel Element Facility at the Australian Atomic Energy Commission to total doses of 2.0 X 105 rads or 1.0 X 106 rads, both at a dose rate of 1.84 X 105 rads/h. After irradiation, the grafted cellulose acetate strips were removed immediately from the monomer solutions and extracted with xylene for 24 h in a Soxhlet. The strips then were dried under vacuum for 24 h at room temperature and weighed. 

Borate buffer, introduced in our laboratory for paper electrophoresis of gastric juice (G19) has since been used for this purpose by other authors (BI2, C5a, Dl, FI, G33, G34, HI, K2, Via, Wll) and also for electrophoresis on cellulose acetate strips (P3, P4). Instead of vertical paper electrophoresis units, some of these investigators used borate buffers with horizontal units (B13, FI, K2, Via). The resolution obtained was certainly less satisfactory than that yielded by the vertical unit (Via) and cathodic peaks were not resolved (B13, C5a, K2). 

Piper et al. (P3, P4) applied the paper electrophoretic method of our laboratory to gastric juice electrophoresis on cellulose acetate strips. They collected gastric juice after augmented histamine stimulation, but gastric acidity prior to collection was neutralized in situ by intragastric instillation of sodium bicarbonate in 5-20% solution. 

Figure 11.5 Migration patterns of a mixture of five Hb species (a) by IEF over the pH 6-9 range, and (b) by electrophoresis on cellulose acetate strips at alkaline pH.8 [Reprinted, with permission, from P. Basset, F. Braconnier and J. Rosa, J. Chromatogr. 227, 1982, 267-304. An Update on Electrophoretic and Chromatographic Methods in the Diagnosis of Hemoglobinopathies . 1982 Elsevier Publishing Company.]...

Electrophoresis is used to study and measure the protein content of biological fluids (see Chapter 5). Types include serum protein electrophoresis using either cellulose acetate strip or agarose gel as the separation media, capillary electrophoresis (CE), immunofixation, and Western blotting. ... 

Specific antisera may be applied with saturated filter paper or cellulose acetate strips, but many commercial kits use other application methods such as mylar templates that allow direct pipetting of antiserum onto the surface of the gel. 

Farrelly and Watkins (F4) have used high voltage electrophoresis of unmodified urine or deproteinized serum for the rapid separation of fourteen amino acids in one direction on a thin-layer plate. Evered and Dando (E16) have employed low voltage electrophoresis for one way separation of amino acids on Whatman No. 1 paper using various buffer solutions. They stated that only the acidic and basic amino acids, p-amino acids, and cystine could be separated completely from a complex mixture such as blood or urine. Scherr (SIO) and Stevens (S52) have also used low voltage electrophoresis for the unidirectional separation of amino acid mixtures on cellulose acetate strips. 

Seherr, G. H., Use of cellulose acetate strips for electrophoresis of amino acids. Anal. Chem. 34, 777 (1962). 

A3. Albrecht-Recht, F., Quantitation of plasma proteins on cellulose acetate strips. Clin. Chim, Acta 4, 627-638 (1959). 

ITP can be carried out preparatively using such supports as polyacrylamide or Sephadex. This method will be discussed further in Sections 2.4.4 and 2.4,5. On a microscale, both commercially available instruments can operate in a semipreparative mode. The LKB instrument, the Tachofrac, uses a cellulose acetate strip to collect the separated sample (A15, A16, MIO), while the Shimadzu equipment relies on the manual removal of the zone of interest by means of a microsyringe. The use of the available equipment is described in Section 2.4. A detailed discussion of equipment and instrument design is to be found in the book on ITP by Everaerts and co-workers (E7). 

Serum proteins have been isolated by preparative ITP. Several stabilizing media have been used, e.g., polyacrylamide (B20, K15) and Sepha-dex (B7, B8). Preparative capillary ITP has been used for the separation of microgram amounts of some human serum proteins (MIO). The separated protein zones were collected on the Tachofrac cellulose acetate strip and could then be further analyzed by Immunoelectrophoresis. 

In 1957, Kohn (K2, K3, K4) demonstrated that cellulose acetate strips have excellent qualities for ImEl. Pore size is 0.5-3.0 p, and the stained strips can be made completely transparent by immersion in white oil. They are somewhat easier to handle than agar gel, but lipoproteins do not stain well, as the background cannot be washed out adequately. Tailing is not encouraged, as the run lasts only 2 hours. 

These disadvantages are not important when strips of 12 X 2.5 cm are used. In this case, with only 1 to 3 (d of serum on a 1.5-cm streak, best results are obtained. It is even possible to use as little as 0.1 of serum (K5). Cellulose acetate strips may be scanned after clearing by immersion in a nonvolatile fluid of refractive index of about 1.47, when they become completely transparent (see, however. Section... 

Cut from a roll or obtain 3 cellulose acetate strips about 20 cm long and 35 mm wide. Lay them on the table top with the curved ends upward. 

Use a syringe and place a small drop of that dye mixture to the side along one edge of the cellulose acetate strip at the same place that you spotted your sample. Poke it into the gel, but keep the area small. This dyes the albumin in the blood serum. The albumin is the fastest moving component, and the dye is used to tell you how far the separation has progressed. Usually, two spots are formed use the darkest one. 

Cellulose acetate strips have some other distinct advantages over paper strips as support media in electrophoresis. These strips greatly improve electrophoretic separations of polar molecules. Cellulose acetate may be cleared more easily than paper to produce transparent strips for automatic scanning. Further, cellulose acetate strips are thinner, more homogenous and chemically purer than paper strips. 

Ing cellulose acetate strip. This Instrument Is marketed by LKB Pro-dukter under the name Tachofrac. Kobayashl et al. (30) have also described preparative capillary ITP experiments using a syringe to extract fractions after migration across a potential gradient detector. Hjert n (31) has described a micropreparative zone electrophoretic technique In which the separation takes place In a capillary filled with a polyacrylamide gel. A flow of buffer past the end of the capillary sweeps the samples through a UV detector after which collection Is possible. This last method would seem to be the least useful because the smaller diameter tube limits the protein capacity to the ng to ug range. 

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