Sephacryl protein separations

For the purpose of high-resolution fractionation, the gel medium must be tailor made to cope with different separation ranges. The Superdex family is designed for the high resolution of peptides and proteins having a molecular mass of 500 to 100,000. Also, Sephacryl media have found wide applicability as a final polishing step in process scale SEC (see Section III,C). 

FIGURE 2.15 Influence of the pore size of Sephacryl HR on the separation of proteins of various molecular mass. The protein mixture is composed of ferritin, aldolase, ovalbumin, and chymotrypsinogen A. [Reproduced from Hagel et al. (1989), with permission.]... 

Figure 1. Separation profile on Sephacryl SlOO column of extracellular PG. 2.7 ml fractions were analyst for protein ( ) and reducing sugars released (A). Peaks I, II, III and IV correspond to PG activity expressed as pmol galacturonic acid released min V...

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). 

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 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). 

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. 1. Purification of myristoylated Arfl. (A) Elution profile of Arfl proteins on Sephacryl S-100 26/60 column. The peak containing Arfl is labeled. Inset shows the expression of myristoylated Arfl in E. coli with NMT. Lane U, uninduced lane 1, induced., band containing Arfl. Proteins from the indicated elution fractions were separated by SDS-PAGE in the gel shown below the chromatogram. marks the band containing Arfl. (B) Resolution of myristoylated ARFI on Phenyl Sepharose HP column. The protein is loaded in the presence of 3 M NaCl and eluted with a descending sodium chloride gradient. In the SDS-PAGE fractionation of the proteins from the elution, shown below the chromatogram, the band containing Arfl is marked with. ...

The purification procedures applied are given in Table 1. Especially gel chromatography on Sephacryl S-300 superfine shortened the process of purification including a well performed separation of the ATPase protein from the adenylate kinase. 

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