Linear polyacrylamide

Radko, SP Chrambach, A, Electrophoretic Migration of Submicron Polystyrene Latex Spheres in Solutions of Linear Polyacrylamide, Macromolecules 32, 2617, 1999. 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

One of the most impressive examples for the detection of a transcription factor was published in 1996, when a mobility-shift assay in a linear-polyacrylamide-filled capillary using fluorescein-labeled DNA showed 100 times higher sensitivity than the conventional slab-gel technique with 32P. Furthermore, the detection of a transcription factor in a single sea urchin egg was demonstrated (24). 

A Freeman, Y Aharonowitz. Immobilization of microbial cells in crosslinked, polymerized linear polyacrylamide gels antibiotic production by immobilized Streptomyces clavuligerus cells. Biotechnol Bioeng 23 2747-2759, 1981. 

The mechanism of separation with linear polymers is as follows. At a certain polymer concentration known as the entanglement threshold, the individual polymer strands begin to interact with each other, leading to a meshlike structure within the capillary. This allows DNA separation to take place. Many of the common polymers are cellulose derivatives, such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and methylcellulose. Other applicable polymers include linear polyacrylamide, polyethylene oxide, agarose, polyvinyl pyrrolidone, and poly-N. Ar-dimethylacrylamide. High-resolution separation up to 12,000 bp has been reported using entangled polymer solutions. 

FIGURE 3.4 CCD images of 100 pM rhodamine B in water obtained at the intersection of a side-arm channel with a main channel on a microchip, applying a positive potential at the top channel relative to the side arm and using (a) all native glass surfaces, and (b) a native glass main channel and a linear polyacrylamide surface coated side-arm channel [816]. Reprinted with permission from the American Chemical Society. 

There are several ways to reduce or suppress the electroosmotic flow in capillaries. These methods involve either eliminating the zeta potential across the solution-solid interface or increasing the viscosity at this interface. One approach is to coat the capillary wall, physically, with a polymer such as methylcellulose or linear polyacrylamide. Because of the difficulty in deactivating the capillary surface reproducibly, however, alternative methods employing dynamic reduction of solute-capillary interactions have been developed. Dynamic reduction of these interactions include the addition of chemical reagents such as methylhydroxyethylcellulose, S-benzylthiouro-nium chloride, and Triton X-100. 

Another successful example is the separation of a series of steroids listed in Fig. 6.11 using a monolithic capillary column prepared by redox initiated polymerization of a solution of acrylamide 4, methylene bisacrylamide 5, vinylsulfonic acid 12, and dodecyl acrylate 18 in N-methylformamide/TRIS-boric acid buffer (pH 8.2) to which polyethylene glycol) (MW 10,000) was added (overall composition 5% T, 60% C, 10% vinylsulfonic acid, 15% lauryl acrylate, 3% polyethylene glycol)). The capillary tube was first vinylized and its part beyond the detection window was coated with linear polyacrylamide to avoid band broadening. Since laser induced fluorescence was used to decrease the detection limit of the method to about 100 attomoles for neutral steroids, all of the analytes were first tagged with dansylhydrazine. Fig. 6.12 shows an... 

Commercially available linear polymers and CGE kits are available. The chemical composition of some of these polymers is proprietary but others are not. Companies marketing these linear polymers for use in CGE include Bio-Rad,84 Perkin Elmer/ABI (linear polyacrylamide),85 and Beckman Instruments (PEO).86 The Beckman SDS-Protein Gel Column Kit includes many of the supplies and reagents required for Protocol 9. 

Acrylates. Acrylamines are widely used to create polymeric coatings on fused silica capillaries in a two- to three-step process. Linear polyacrylamide is then grafted to the appropriately prepared surface. The problem of acrylamide stability can be solved by using a monomer that is inherently more hydrolytically stable. 

Hb E, Hb C, and Hb G have been distinguished by their altered mobility in CZE under denaturing conditions using linear polyacrylamide-coated capillaries.36,37 Hemoglobins are denatured and separated in 10 mM phosphate buffer (pH 2.5) using 7 M urea and 0.1% Triton X-100... 

Figure 13 Molecular-mass analysis of phosphorylase B (panel B) and the separation of the noncovalently (Sypro Red) labeled protein markers (panel A ALA, a-lactalbumin CBA, carbonic anhydrase OVA, ovalbumin BSA, bovine serum albumin BGA, (f-galactosidase) and the covalently (FITC) labeled protein markers (panel C TRI, tripsin inhibitor CAH, carbonic anhydrase ADH, alcohol dehydrogenase BSA, bovine serum albumin BGA, P-galactosidase). Separation conditions gel, 1% agarose, 2% linear polyacrylamide (LPA, MW 700,000-1,000,000) in 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation buffer, 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation voltage, 420 V, current, 5 mA gel thickness, 190 pm effective separation length, 3.5 cm temperature, 25°C sample loading, 0.2 pL into 2.5 x 4 x 0.19-mm injection wells. Sample buffer contained 0.05% SDS and 1 x Sypro Red. (Reproduced with permission from Ref. 141.)...

On account of their simple preparation and commercial availability there is a wide variety of applications for linear polyacrylamide coatings. These involve not only the separation of proteins but also include biomolecules such as DNA fragments. They are also used for the preparation of gel-filled capillaries. 

---