Modified Sephadex Gels

Chang (64) used a porous styrene/divinylbenzene gel to determine small amounts of fatty acid dimers in tall oil. Hase and Harva (66) separated the monomeracid methyl esters from dimers and higher oligomers using a modified dextran gel SEPHADEX LH-20. 

Purify the sulfhydryl-modified protein by dialysis against 50 mM sodium phosphate, 1 mM EDTA, pH 7.5, or by gel filtration on a Sephadex G-25 column using the same buffer. Again, if a peptide of low molecular weight is being modified, use gel filtration for purification. 

Alumina Silica gel Modified silica gel Kieselguhr Cellulose powder Modified cellulose, e.g. D E A E and C M Sephadex gels Adsorption or partition Adsorption or partition Adsorption, partition Partition Partition Ion-exchange Exclusion... 

E. Sephadex Sephadex layers are prepared from modified dextran gels for the separation of hydrophilic solutes such as nucleic acids and peptides. The mechanism of separation is partition chromatography governed by size exclusion in the swollen gel containing pores of controlled dimensions. The gels, a layer spreader and special equipment for developing layers are available from Pharmacia Fine Chemicals. 

In addition to the classical column packing materials (silica gel, alumina, celite), other widely used columns are of the polydextran gel type for the fractionation of the polar steroids, Sephadex LH-20, and for the less polar steroid compounds its hydro-xyalkyl derivative, Lipidex. The use of ion exchange columns (e.g., DEAE-Sephadex A-25, Amberlite XAD-2) is also widespread. High selectivity is attainable with immunoaffinity chromatography, where antibodies are immobilized on chemically modified agarose gel and filled in columns. For the solid-phase... 

The CLU also modify the gel structure, making it less "sugar-like". A detailed fragmental analysis (19) of a more highly cross-linked Sephadex gel (G-25 -see Table I) revealed that more than a third of the CLU were present as mono ether linked pendent side-arms and of the rest most were coupled to two ether... 

The starting protein (bovine RNase) is chromatographed on a G-16 Sephadex gel column with 1 mM HCl or 5 mM phosphoric acid as the eluent. The concentration of the eluted RNase, approximately 2 mg/ml, is determined using the absorbance coefficient of 7.3 at 280 nm for a 1% solution. The perturbed protein is reacted with a modifier, such as HMPA, for 15 minutes and the pH titrated to 8.5 with 0.05 M NaOH. The initial activity of the modified protein solution is measured at pH 8.5 prior to crosslinking. 

Purify the modified liposomes from reaction by-products by dialysis or gel filtration using a column of Sephadex G-50. 

The modified liposomes may be separated from excess protein by gel filtration using Sephadex G-75 or by centrifugal floatation in a polymer gradient (Derksen and Scherphof, 1985). 

A lipophilic gel, namely, Sephadex LH-20, has found application in studies of partially acetylated dextrans. In describing a modified procedure for the replacement of the O-acetyl groups in such dextrans by O-methyl groups prior to acid hydrolysis and identification of the fragments (which indicates the distribution of the substituents in... 

The gel-like, bead nature of wet Sephadex (a modified dextran) enables small molecules such as inorganic salts to diffuse freely into it while, at the same time, protein molecules are unable to do so. Hence, passage through a Sephadex column can be used for complete removal of salts from protein solutions. Polysaccharides can be freed from monosaccharides and other small molecules because of their differential retardation. Similarly, amino acids can be separated from proteins and large peptides. 

Separate excess cystamine and EDC (and reaction by-products) from the modified protein by dialysis or gel filtration using 10 mM sodium phosphate, 0.15 M NaCl, pH 7.2. A desalting column may be used for the gel filtration procedure (i.e., Sephadex G-25 from Pharmacia). 

Quickly isolate the modified protein by gel filtration using Sephadex G-25 or the equivalent. 

Purify the derivative by gel filtration using a PBS buffer or another suitable buffer for the particular protein being modified. The use of Sephadex G-25 or similar matrices with low exclusion limits work well. To obtain complete separation, the column size should be 15—20 times the size of the applied sample. Fluorescent molecules often nonspecifically stick to the gel filtration support, so reuse of the column is not recommended. 

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