Sephadex G-50 columns

Fig. 2.155. Gel filtration chromatography of MSW in a Sephadex G-50 column. Solid line represents 10 per cent diluted MSW decolourized by the live immobilized fungus and the broken line represents fractionation of untreated, 10 per cent diluted MSW. The colour absorbance of the fractions was read at 475 nm. MSW treated with the fungus for five days was used for gel filtration. Reprinted with permission from C. Raghukumar et al. [327].

Using pyranine (8-hydroxy-1,3,6-pyrene trisulfonate) as intraliposome pH indicator, the liposomes were prepared as above (as in section Preparation of 100 nm SSL Loaded with DOX via Transmembrane AS Gradient ) with the exception that pyranine (0.5 mM) was included in the hydration solution. Removal of untrapped pyranine was achieved by gel filtration on a Sephadex G-50 column, preequilibrated with either NaCl, KCl, sucrose or AS solution (according to need). All these solutions also contained lOmM Hepes buffer at the desired pH (usually pH 7.5). 

Since the glucose-lysine reaction mixture used in this study consisted of a number of different substances, it was of interest to study whether the observed inhibitory effect in vitro could be attributed to some specific compound(s). In order to obtain a separation, an aliquot of the LMW fraction, radiolabel led by [U- glucose added to the reactants, was applied on a Sephadex G-50 column and eluted with water. The UV absorbance was recorded and the eluate was collected in fractions. The degree of inhibition effected by small samples of equal volume from each fraction and exerted on carboxypeptidase A and purified aminopeptidase N was determined as well as the radioactivity... 

Fig. 1.4 Chromatographic separation of tryptic peptides from m-AspAT. A. Sephadex G-50 column chromatography. Fractions I—111 were applied on DEAE-cellulose chromatography (B). B. DE-52 column chromatographies of fractions, I, II and III. Data are from Kagamiyama et al.2S)...

Separate unincorporated nucleotides from the labeled probe with a Sephadex G-50 column (using a homemade apparatus17) or a commercial device designed for this purpose. 

Heterologous Hybridization Procedure. The DNA fragment to be labeled is denatured in a small volume of distilled water (up to 12 fA) at 100° for 10 min, quick-cooled on ice, and spun down to collect the condensate. Set up a labeling reaction with 2 fA of 10 X primer buffer, 3 /A of dNTP mix, 2 fA (20 ftCi) of [a-32P]dCTP (3000 G/mmol, aqueous solution), and 12 fA of denatured DNA, on ice. Start the reaction by adding 1 /A of 2 U /fA Klenow enzyme (DNA polymerase I, large fragment) and incubate at 37° for 1 hr. Stop the reaction by adding 2 fA of 0.2 M EDTA (pH 8.0). It is optional to separate the labeled probe from unincorporated nucleotides, for example, on a 1-ml Sephadex G-50 column. 

Urea affects the gel as well as the state of aggregation of solutes. Stepa-now et al. (1961) have shown that formation of peptide-peptide complexes may be avoided in phenolate or alkaline urea solutions. Two peptides derived from tobacco mosaic virus protein could be separated with Sephadex only in the presence of 8 M urea. Urea need only be included in the sample solution, not in the eluting solvent (0.01 M sodium hydroxide). Upon filtration on a Sephadex G-50 column, urea and the two peptides moved as well-separated zones. The authors did not comment on the choice of G-.50. It was probably found to be superior to G-25 since urea seems to close the pores and meshes of a Sephadex gel. The reduction in effective pore and mesh size is perhaps caused by urea being bound to the carbohydrate network since the swelling is in fact increased in strong urea solutions. 

Prepare Sephadex G-50 column equilibrated with PBS such that the packed volume is at least six times the volume of conjugate to be applied. Allow a disc of filter paper, cut to fit the dimensions of the column, to float onto the top of the column. This facilitates the even application of conjugate. 

Sephadex G-50 column. Add 20 ml of phosphate buffer to 1 g of Se-phadex G-50 Fine. Let stand at least 3 hr at room temperature, then pour the slurry into a 10-ml disposable glass serological pipette containing a small amount of glass wool at the tip. The bed volume should be about 10 ml. 

Wait 5-30 min, then stop the reaction by adding phosphate buffer containing 0.1% azide. Transfer the iodination reaction to a Sephadex G-50 column and wash the reaction vial with 5% BSA-phosphate buffer, transfering the washes to the column. Start the column flow after all the washes and transfers have been completed. Collect 0.4-ml fractions in each of 20 tubes. 

The encapsulation efficiency of a thalidasine liposome injection was studied using Sephadex G-50 column fluorimetry. The free alkaloid was separated from the liposome particles by the column, with satisfactory recovery of the liposome particles, as well as column reproducibility. The encapsulation efficiency of this particular liposome preparation (139-2) was approximately 93% due to the highly lipophilic property of the alkaloid. The authors concluded that the method was suitable for the quality control of encapsulation efficiency of this polyphase liposome injection [147]. 

Pass the solution down a Sephadex G-50 column (10 mL gel/mL of solution) equilibrated in HBS (pH 7.4). Collect and combine fractions with an absorbance >1.0 at 280 nm. In cases where the yield is low it may be necessary to collect fractions with lower absorbances as well. 

DTPA-succinylated polylysine (DTPA-PL) of various molecular weights is separated from free DTPA and free succinic acid by column chromatography on a 1.5 x 45 cm Sephadex G-50 column. The modified polylysine is eluted in the void volume, whereas the free DTPA and succinic acids are eluted in the salt volume. The column is equilibrated and developed in deionized distilled water. The elution is monitored by spectrophotometric reading at 230 nm. 

Remove excess periodate by gel filtration on a Sephadex G-50 column (20 x 1.5 cm) using borate-buffered saline (20 mM borate containing 120 mM NaCl, pH 8.4) as the eluent. 

Separate free drug from the liposomized drug by gel filtration over Sephadex G-50 column. 

The level of the lx HSS buffer volume on the Sephadex G-50 column prepared in step 12 of Subheading 2.1 is reduced to just below the level of the Sephadex. Do not allow the... 

In some experiments vesicle membranes treated with phospholipase A and control vesicles were separated from the ACh in the suspension medium by gelfiltration on a Sephadex G-50 column. Vesicles or enzyme-treated vesicles were identified by protein analysis and came out with the void volume. After acid boiling it was found by bioassay that the enzyme-treated vesicles contained less ACh than the controls. 

Purified oligonucleotides (80-100 nmol) were dissolved in 0.1 mL of 20 mM sodium acetate (pH 4.4) and 4.6 /jumol of sodium metaperiodate was added. The reaction was stirred for 30 min at 0°C in the dark. An equal volume of poly-Lys (Sigma, mean 15 kd) 80-100 nmol in 2 M NaCl, 0.2 M sodium borate buffer (pH 8.4), and 100 /miol sodium cyanoborohydride were added. The mixture was incubated overnight at 20°C and then loaded on a Sephadex G-50 column equilibrated with 0.5 M NaCl, 20 mM sodium acetate buffer (pH 6.0). Each fraction was assayed for its oligonucleotide-poly-Lys content by absorbance at 260 nm and by protein assay. The conjugates were stored at — 80°C. 

Figure 6. The distribution of metals between MT and the low molecular weight fraction after incubation of NotAuTM and equine renal MT at a lO/L Au/Cd ratio for a, 2 h and b, 26 h. The reaction is complete within 2 h. Conditions Sephadex G-50 column (1.5 X 60 cm) eluted with Tris-HCl 40mM, pH 7.8 at 5 mL/h 3.2 mL fractions.

Unless indicated otherwise, partially purified [5j soya lecithin (Sigma, type IV-S) was used. FOFl was isolated from R. rubrum as described [l]. Proteoliposomes were formed by incubation of FOFl in the presence of lipid and cholate, followed by centrfugation through Sephadex G-50 columns in order to remove the cholate. A molecular weight of 545 kDalton was... 

Prepare a spun Sephadex G-50 column. Block the end of a 1-mL disposable syringe with glass wool. Pill with Sephadex G-50 nntil all the excess fluid drains out and the beads are packed. Position the colnmn in a disposable 15-mL plastic conical centrifuge tube, and centrifuge at 4,000g for 3 min. Equilibrate this column with 100pL MITE by spinning as before. Do this five times. 

Pass the eluted DNA (maximum volume 100 pL) through the equilibrated Sephadex G-50 column, collecting the purified DNA in a fresh 1.5-mL Eppendorf tube (with the lid cut ofl) placed at the bottom of a fresh 15-mL centrifuge tube. 

The molecular weight distribution was determined using a chromatographic system with a 57 X 1.8 cm Sephadex G-50 column in 0.5 mol. L NaOH. The mobile phase was 0.5 mol. L NaOH, and the flow rate was 0.4 mL.min. Fractions of 4 mL were collected, and the absorbance of each fraction was read at 280 nm. Sample volumes of 0.4 mL at 2.0 mg/mL were injected. The chromatographic column was previously calibrated with proteins of known molecular weight. 

NaDDS + NaTDS, were studied on Sephadex G-50 column for different ratios of (NaTDS/NaDDS) and with varying total concentration [52,b] The void volume of the column was determined by using Blue Dextran 2000. The retention volumes were determined for samples of mixed surfactant solution of volume 50 ml. The elution curves for mixed NaDDS NaTDS, 1 1 molar ratio, are given as a function of total concentration, C, in Fig. 20. At concentrations of below cmc, the curves are sigmoidal, and the retention volumes are independent... 

---