Exclusion limit sephadex gels

Exclusion Limit This is defined as the molecular mass of the smallest molecule that cannot diffuse into the. inner volume of the gel matrix. All molecules above this limit elute rapidly in a single zone. The exclusion limit of a typical gel, Sephadex G-50, is 30,000 daltons. All solute molecules having a molecular size greater than this value would pass directly through the column bed without entering the gel pores. 

Briefly describe how you would experimentally measure the exclusion limit for a Sephadex gel whose bottle has lost its label. 

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. 

Gel filtration media such as Biogel P-30 (fine or medium) or Biogel P-6 (fine or medium Bio-Rad Laboratories Hercules, CA) Sephadex G-10, G-15, or G-25 (Amersham Biosciences Piscataway, NJ) or equivalent matrices made by many other manufacturers are typically used. For optimum separation of the biotinylated antibody from unattached label, researchers should choose a gel matrix with an exclusion limit smaller than the molecular weight of the protein sample. Thus, the protein will elute in the void volume of the column while smaller unwanted components will be strongly retarded by the matrix. Neither biotin nor antibody molecules are colored, so the progress of the gel filtration column must be monitored by absorbance at 280 nm. Small quantities of biotinylated antibodies can be... 

Mercapto- 5-SH)-polycy tidy lie acid has been shown by Bardos et al.55 to block the DNA-directed RNA synthesis at 1/2 to 1/50 of the concentration of the unmodified DNA template used in the reaction. This mercapto derivative of polycytidylic acid (MPC) has been prepared by partial thiolation of polycytidylic acid (PC) according to the general procedure of Bardos et al.56> 57> the thiolated compound was gel-filtered through a Sephadex column and subsequently, through an Agarose-1.5 m (Bio-Gel A-l.5 m, exclusion limit 1,5000.000 mol. wt., Bio-Rad Labs.) column, then lyophilized and redissolved in 0.1 M Tris-buffer58). This compound (conversion of 9.5% of the cytidylate units to 5-mercaptocytidylate) was studied in the DNA-polymerase system of oncorna viruses. 

TABLE 2. Range of Sephadex and Sepharose Gels Manufactured by Pharmacia Showing Their Fractionation Range and Exclusion Limit... 

The free and complexed Cd (II) are separated by two 25 cm HPLC columns of Sephadex G-10 (a cross-linked dextran gel of 40-120 p bead diameter). The mobile phase was distilled deionized water. Sephadex G-10 xerogel has an exclusion limit 700, that is, it can be used to fractionate species of molecular weight less than 700. The larger Cd-fulvic acid complex is unretained and elutes before hydrated Cd (II). As with the phosphorus esters above, SEC is a viable method not only for separating these complexes for analysis but also for purification. 

Reverse phase HPLC was successful in separating the bulk of the monomeric material from DHE with the exception of protoporphyrin. This component was present in the broad elution profile of the active fraction and could not be separated from HPD by reverse phase HPLC (Figure 4.13). However, the active component could be isolated quantitatively by gel filtration using aqueous elution on a Sephadex (G25) or a poly-aerylamide column (nominal exclusion limit 20000 Da). Non-aqueous fractionation using a tetrahydrofuran/methanol/aqueous buffer eluant on Sephadex-LH20 has also allowed the isolation of the active component from the above mixture free of any monomeric species [43]. This column material, -LH20, has also been used to successfully remove excess cuprous acetate present in the preparations of copper amine derivatives of protoporphyrin [44]. 

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