Sephacryl column chromatography

In the process of determining the molecular weights of the polymers by Sephacryl column chromatography, it was found that several of the polymers produced two peaks in the elution profile. This is illustrated in Figure 8. This result indicates the existence of two different size classes of polymer chains within the same polymer preparation. Furthermore, the number of peaks present in the elution profile and their relative proportions varied among different batches of the same polymer... 

Table 5. Molecular Weights from Sephacryl Column Chromatography... 

The xylan from the silk floss (PI) was homogeneous on gel-column chromatography over Sephacryl S-300, having a molecular mass of 52000. 

Instrumentation. A Pharmacia BioPilot Column Chromatography system was used to perform large-scale size exclusion chromatography (SEC) with an 11.3 x 90 cm BioProcess column packed with Sephacryl S-200 HR gel. High performance size exclusion (HPSEC) and ion exchange chromatography (HPIEC) were conducted with Pharmacia Superose 6 and 12 (HR 10/30) and Mono-Q (HR 5/5) columns respectively, equipped with Beckman model 520 system controller and Beckman model HOB HPLC pumps. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Column chromatography apparatus, including a suitable low-pressure pump, on-line UV monitoring at 280 nm and a fraction collector. Chromatography media Sephacryl S-200 (SF), Sephadex G-25 (SF) (Pharmacia). 

The enzyme was purified 178-folds with 90-95% homogeneity and showed a specific activity of 7 umol NADH oxidised min—1 mg—1 protein by emplying ion-exchange (DEAE cellulose. Mono Q), gel filteration (Sephacryl S-300) and two affinity column chromatography. The best separation of the enzyme protein was obtained with nucleotide analogue affinity ligand reactive-red 120 agarose. The enzyme... 

Fig. 6-11. Column chromatography of (a) post-SE53 IgGj peak and (b) L243 tissue culture fluid on sephacryl S-300 using 0.5 M-NaCl at laboratory scale (1.5 cm i.d.x90cm) at a flow rate of 1 mL min. ...

Extraction and purification of luciferin and luciferase (Viviani etal., 2002a) To isolate luciferin, the lanterns of the Australian A. flava were homogenized in hot 0.1 M citrate buffer, pH 5, and the mixture was heated to 95°C for 5 min. The mixture was acidified to pH 2.5-3.0 with HCl, and luciferin was extracted with ethyl acetate. Upon thin-layer chromatography (ethanol-ethyl acetate-water, 5 3 2 or 3 5 2), the active fraction of luciferin was fluorescent in purple (emission Lav 415 nm when excited at 290 nm). To isolate the luciferase, the cold-water extract prepared according to Wood (1993 see above) was chromatographed on a column of Sephacryl S-300. On the same... 

Figure 30 shows the results of an experiment in which a solution of Q-CdS was fractionated by exclusion chromatography in a column of sephacryl-gel This column material has holes which the smaller particles penetrate and reside in for some time. The first fraction therefore contains the larger particles. The upper part of the figure shows the absorption spectrum of the starting material, and the lower part the spectra of six fractions. The first fraction has an unstructured spectrum beginning at... 

Purify the conjugate from unreacted protein or unreacted dendrimer using gel filtration chromatography with a matrix having an exclusion limit appropriate to accommodate the size of the molecules being separated (i.e., a HiPrep 16/60 column packed with Sephacryl S-200 HR, GE Healthcare). 

Three extracellular P-mannanases (M-1, M-II and M-III) were purifled by anunonium sulfate precipitation (80% saturation) followed by chromatography on a DEAE-Toyopearl 650 M column (4.6 x 35 cm) equilibrated and eluted with 0.01 M phosphate buffer (pH 7.0 ) and by a hydroxylapatite column (1.6 x 25 cm). As shown in Fig. 1, two active fractions (Fraction 1 and 2) were detected after hydroxylapatite chromatography. Each fraction 1 and 2 was applied onto a Sephacryl S-200 column (2.6 x 90 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1 M NaCl and eluted with the same buffer. Mannanase-I and -II were isolated from fraction 1 and mannanase-III was from fraction 2. 

Estimation of Polymer Sizes by Gel Permeation Chromatography. The copolymer (1 mg) was dissolved in 1 ml of phosphate buffered saline (PBS), pH 7.4, and applied to a column of Sephacryl S-300 (1 X 108 cm) or Sephacryl S-400 (1 x 114 cm). The column was eluted with PBS at a flow rate of 0.2 ml/min. The elution profile of the copolmer was monitored by its absorbance at 214 nm. Bovine serum albumin (BSA) was chromatographed for comparative purposes and polyacrylamide standards (Modchrom, Inc.) were used. 

Gel Permeation Chromatography The relative molecular weights of the copolymers were compared by chromatography on a 1 0 x 116 cm Sephacryl S-400 column with detection by absorbance at 214 nm. This also enabled the determination of the relative efficiency of polymerization reactions via integration of the total column volume (Vt) peak. This was possible because the monomers are the predominant species of small molecules that absorb at 214 nm. 

Fig. 1. Gel permeation chromatography of an antibody-toxin conjugate reaction mixture. The reaction mixture obtained following the conjugation of a mouse monoclonal antibody (50 mg) and [l25I]-labeled abrin A chain was chromatographed on a column of Sephacryl S-200 (SF), dimensions 80 cm x 2.6 cm (id). Fractions eluting from the column were monitored spectrophotometrically at 280 nm (—) to measure total protein, and by gamma counting (—) to measure the A chain in its free or conjugated form. The hatched area indicates a typical pooled conjugate preparation.

Equilibrate a Sephacryl S-200 HR chromatography column with chromatography elution buffer (3 column volumes) using a peristaltic pump. 

Fig. 1. Absorption spectra of the different fractions obtained during the purification of PSI-enriched particles of P. laminosum. The spectra were recorded at room temperature on a Beekman UV 5260 spectrophotometer using 1 cm path length quartz cuvettes, (a) Sonicate (—) 40,000xg supernatant (...) DEAE-cellulose eluate (—). (b) PSI-enriched particles after chromatography on a Sephacryl S-300 column.

Separate unreacted enzyme from the mixture by chromatography on a column of Sephacryl S-200 equilibrated with PBS or by salt precipitation with ammonium sulfate as described above. 

Figure 9. Gel filtration chromatography of the HCl-soluble apricot pectin fraction, degraded by various enzyme systems on a Sephacryl S—300 column (68 x 1.05 cm), eluent 0.05 M phosphate buffer pH 7. AGA is anhydrogalacturonic acid Pectinex is a wide-spectrum commercial pectinase. (Reproduced with permission from ref. 54. Copyright 1985.)...

M NaCl at pH 5.5 to remove most of the FG II and other soluble components. The insoluble fraction was then extracted with 1.2 M NaCl at pH 6. The extract was concentrated by ultrafiltration, adjusted to pH 5, and heated 7 min. at 100 C to inactivate the polygalacturonases. The FG converter in the heated solution was then purified by chromatography on Sephacryl S-200 and Folyanion SI and by chromatofocusing on a Mono F column. A 28-fold increase in the specific activity of the converter was achieved with an 81% recovery of total activity (Table I). 

Arabinogalactan proteins (AGP) represented a major proportion (40%) of the total soluble polysaccharides (300 mg/1) in a red Carignan wine (Pellerin etal., 1995 Vidal etal., 2001). These macromolecules were first fractionated on a DEAF Sephacel anion exchanger, then separated from the yeast mannoproteins by affinity chromatography on Concanavaline A and finally purified, by exclusion chromatography on a Sephacryl S400 column. Table 3.4 shows the molecular characteristics of the five species obtained. 

The enzyme preparations obtained were concentrated and equilibrated with Tris-HCl buffer (50 mM, pH 8) by membrane filtration using an Amicon celt with a PM 10 membrane and fractionated on a Q-Sepharose (Pharmacia LKB) column (26 x 400 mm). The column was previously equilibrated with the same buffer. Proteins were eluted with a linear gradient of NaCl, at a flow rate of 250 ml.h l, and the eluate was collected in 10 ml fractions. Active fractions were pooled and concentrated in an Amicon cell. The resulting enzyme solution was applied to a Sephacryl S-300 (Pharmacia LKB) chromatography gel filtration column (10 x 1000 mm) equilibrated with Tris-HCl buffer (50 mM, pH 7). The elution rate was 19.5 ml.h and the eluate was collected in 1.5 ml fractions. All the fractions were also analyzed for P-glucosidase activity and protein concentration. In the case of a non pure P-glucosidase preparation, fractions from the previous step were applied to a Hydroxylapatite (Biorad) column (16 x 400 mm) equilibrated with phosphate buffer (6.25 mM, pH 6.8). The elution rate was 50 ml.h" and the eluate was collected in 6 ml fractions. The elution was performed with a linear gradient of phosphate. 

The initial PIA purification method was developed by Mack et al. (3). These authors used a different, two-step chromatography protocol involving size-exclusion and ion exchange chromatography on Sephadex G-200, Q-Sepharose, and S-Sepharose. A similar purification method has been described recently to isolate a PIA-related polysaccharide polymer in E. coli (7). Briefly, E. coli cells were incubated in 50 raM Tris-HCL buffer (pH 8.0), 100 mg lysozyme, and 0.1 M EDTA at room temperature for 2 h. Phenol/chloroform extraction steps were performed to separate protein and debris contamination from the polysaccharide. Samples were concentrated by ultrafiltration devices (10,000 MW cut off) and fractionated on a fast protein liquid chromatography (FPLC) system with a Sephacryl S-2000 column (equilibration and elution buffer 0.1 MPBS, pH 7.4). 

Anaplasma phagocytophilum has also been purified using a packed sterile Sephacryl S-1000 (Pharmacia, Uppsala, Sweden) chromatography column (22) or Percoll (18,23). 

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