Agarose degradation

M. Malmqvist, Purification and characterization of two different agarose-degrading enzymes, Bio-chim. Biophys. Acta, 537 (1978) 31 -3. 

Small-angle X-ray scattering (SAXS), circular dichroism (CD), and UV spectroscopy at different temperatures were used to investigate the nature of calf-thymus DNA in aqueous solution, in the presence of [Me Sn] " (n = 1-3) species. The results demonstrate that the [MeSn(IV)] moiety does not influence the structure and conformation of the DNA double helix, and does not degrade DNA, as indicated by agarose gel electrophoresis. Inter alia, the radii of gyration, Rg, of the cross section of native calf-thymus DNA, determined by SAXS in aqueous solution in the presence of [Me Sn] " (n = 1-3) species are constant and independent of the nature and concentration of the [Me Sn] species. 

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

The purification of the pancreatic alpha amylase was effected on an affinity adsorbent prepared from enzymically degraded starch plus agarose activated with bisoxirane.14 The fractions from the affinity column were analyzed for protein components by u.v. absorbance, and for alpha amylase activity by incubating the fractions with starch and measuring the increase in reducing sugars. The results are shown in Fig. 5. 

Subsurface environmental conditions are suboptimal with low temperatures and low concentrations of growth nutrients. The decline of bacterial inoculae by protozoan predation is of major concern in soil (Acea etal., 1988 Acea Alexander, 1988 Casida, 1989) but may not be a factor in saturated subsurface environments. Immobilization of cells to carrier material may enhance microbial survival in the environment through control of predation and supply of nutrients and moisture. Stormo Crawford (1992) developed a cell immobilization technique for production of small beads (2-50 /rm) consisting of agarose and cells of PCP-degrading Flavobacterium sp. Microencapsulated Flavobacteria efficiently degraded PCP and survived for two years in soil columns at environmental conditions (Stormo Crawford, 1994). These results show that microencapsulation may be a very useful tool in in situ bioremediation. 

If you want to verily that the mRNA is not degraded by trace contamination by RNases, analyze the mRNA by agarose gel electrophoresis using standard protocols (16). Usually, ladder bands or smear bands around lkb-3kb are visible when good mRNA is obtained (see Fig. 4). If RNases are present, some bands smaller than 1 kb are visible. 

Figure 7.7. Agarose gel electrophoresis of total RNA. Total RNA from mouse skin (panel a, lane 2) and two human cadaver skin samples (panel b, lanes 1 and 2) were isolated by guanidine thiocyanate method and size fractionated on denaturing formaldehyde containing 1% agarose gel and stained with 0.5 pg/mL ethidium bromide. Note that in case of mouse skin RNA, two distinct ribosomal RNA bands (upper 28S and lower 18S bands) are clearly visible. In contrast, in case of human skin samples, which were collected several hours postmortem, there is partial RNA degradation as is evident by fuzzy 28S and 18S ribosomal RNA bands. RNA degradation is more pronounced in one of the samples than the other (panel b, compare lane 1 and lane 2). Ribosomal RNA bands are indicated by arrowheads. RNA size markers (Invitrogen, Carlsbad, CA) in the range 0.24 to 9.5 kb are in lane 1 (panel a) and lane 3 (panel b).

M. Malmqvist, Degradation of agarose gels and solutions by bacterial agarase, Carbohydr. Res., 62 (1978) 337-348. 

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