Polypeptides separation

Minakuchi, H., Ishizuka, N., Nakanishi, K., Soga, N., Tanaka, N. (1998a). Performance of an octadecylsilylated continuous porous silica column in polypeptide separation. J. Chromatogr. A 828, 83-90. 

Tanaka, N., Kimata, K., Mikawa, Y., Hosoya, K., Araki, T., Ohtsu, Y., Shiojima, Y., Tsuboi, R., Tsuchiya, H. (1990). Performance of wide-pore silica- and polymer-based packing materials in polypeptide separation the effect of pore size and alkyl chain length. J. Chromatogr. 535, 13-31. 

Polymers and resins Water purification, including removal of phenol, chlorophenols, ketones, alcohols, aromatics, aniline, indene, polynuclear aromatics, nitro- and chlor-aromatics, PCB, pesticides, antibiotics, detergents, emulsifiers, wetting agents, kraftmill effluents, dyestuffs recovery and purification of steroids, amino acids and polypeptides separation of fatty adds from water and toluene separation of aromatics from ahphatics separation of hydroquinone from monomers recovery of proteins and enzymes removal of colours from symps ... 

Fig. 2. Western blotting using a radiolabeled antibody (a) SDS polyacrylamide gel electrophoresis, yielding polypeptides separated into discrete bands (b) nitrocellulose or nylon membrane onto which the protein bands have been blotted (i.e. a Western blot) (c) autoradiograph after incubating the Western blot with radiolabeled antibody, washing away unbound antibody and placing the membrane against X-ray film.

The amount of protein loaded on the gel will depend on several factors—the relative abundance of the protein (s) of interest and the titer and specificity of the antibody, for example. Up to 200 ig/track may be loaded, although at this level, high background on development of the blot may be a problem. The smaller the volume loaded onto the gel, the better the resolution will be in practice, 5-10 pL is an optimal loading volume, although up to 50 pL is possible without completely comprising polypeptide separation. 

The described procedure is the only one that gives a variety of colors which may help the identification of polypeptides separated. It has, however, to be noted that some colors are dependent on the protein concentration. Comparison of the results of different variations of the silver stain method [225,226] and other staining procedures [227,228] proved that the method of Oakley et al. [227] is simple and quickly performed. In the Sammons procedures [224] (see above) the washing steps are rather time consuming but offer superior results. The most complicated procedure is that of Switzer et al. [225]. It must also be noted that the methods of Oakley et al. [227] and Switzer et al. [225] require ammoniacal silver solution which is more difficult to handle than silver nitrate. Also the above described method is the only one that allows more than one gel to be stained at once in the same tray. 

Stoichiometry and copy number of the large subunits present in PS I complexes were also determined by two different methods. The protein content of CPl-a which had been determined by measuring the total amino acid content of the complexes with L-o<-amino-/9-guanidinopropionic acid as an internal standard, was about 250 kDa proteins per P-700. Assuming that each one copy of the 10, 13 and 14 kDa subunits is present in the complex, the value shows that there are two or three copies of the large subunits in the PS I complexes. In the second approach, Synechococcus cells were uniformly labeled with C and radioactivities of the subunit polypeptides separated by SDS gel electrophoresis were determined. Because samples with known P-700 contents were applied to gel, quantities of the subunits estimated from the specific activity of radioactive carbon could be related to the P-700 content. Table II shows that PS I complexes contain one copy each of the two large subunits. Thus, if chlorophyll distributes evenly between the two large subunits, one subunit carry 47 chlorophyll... 

The protein complexes of P. lutherii were separated from thylakoid membranes on a digitonin (0.1%), sucrose gradient (10-40%). Fractions were collected and the polypeptides separated under denaturing conditions on a 15-20% gradient polyacrylamide gel and stained with Silver stain. Polypeptides from other gels were transferred electrophoretically to nitrocellulose membrane and reacted with antisera to the acf complex of P. lutherii. 

Polypeptide separator Ethylene South Africa 4,725 1970 Farbenfabriken Bayer... 

Comparative studies of the separation of chlorophyll-protein complexes either by LiDS-PAGE or digitonin treatment followed by sucrose-density gradient, have led to the isolation of four and three complexes respectively. These complexes have been characterized by their spectral properties and their polypeptides separated. These new data are in agreement with the structural scheme previously proposed. 

---