Sepharose VOLUME

Size exclusion chromatography has been used to analyse the size distribution of liposomes. For example, SUV can be separated from MLV, which elute in the void volume, by using a Sepharose 4B gel. 

The dialysed sample was fractionated by cation exchange chromatography. A 40-50 ml sample was applied to a CM-Sepharose CL-6B column (1.5 x 15 cm). Unbound proteins were removed with 50 mM MES pH 6.8, 1 mM DTT, and the bound proteins were eluted with an increasing NaCl gradient from 0 - 0.4 M NaCl in a total volume of 500 ml. The flow was 25 ml/h and fractions of 8.33 ml were collected. The protein profile was measured at 280 nm. 

One ml of lysis buffer should be added to 10 /il/IP of protein G-Sepharose. The tube should be flicked to mix the contents and then centrifuged at 8000 rpm for 1 min. The supernatant is removed, 500 pi of lysis buffer, 1 to 2 /ig/IP of the relevant antibody are added, and the tube is rotated at 4° for 1 h. The beads are collected after a quick spin and washed twice with 1 ml of lysis buffer. Total cell lysate is added to the beads and the final volume increased to 500 pi with lysis buffer. The tube is rotated at 4° for 1 to 2 h, and the beads are washed twice with 1 ml of lysis buffer containing 0.5 M NaCl before further analysis. To reduce any nonspecific binding, the total lysate can be preabsorbed with protein G Sepharose for 1 h at 4° in order to remove components that bind nonspecifically to the beads. Alternatively, the extract may be incubated with the specific antibody for 1 h at 4°, and the... 

Fractionation by Stepwise Elution. Information obtained from the analytical separation was applied for a preparative purification. Lignin peroxidase concentrate was bound to a Q-Sepharose colunm (0= 5 cm, V = 1000 ml) after ultrafiltration and eluted stepwise with 0.08 M, 0.18 M and 0.28 M sodium acetate, pH 6.0. The fraction which was eluted with 0.28 M buffer (V= 3.91, 4400 U/1) was purified further. It was bound to Q-Sepharose and eluted with 0.18 M and 0.3 M sodium acetate. En rnie in the latter fraction was precipitated and dissolved in glycerol as previously described. The volume was 15 ml. 

These results indicate that there is no competition between NjPjAZg and EtdBr and suggest that NjPjAz does not interact with DNA through an intercalation process but through covalent binding. This is supported by the fact that there is no modification of the fluorescence decrease after 10 dialyses against 200 volumes of 10 M NaClO. Furthermore, it is possible to separate on a gel column (Sepharose 6B) the NjPjA which has reacted with DNA from the free compound and in that way to follow the kinetics of the complexation (data available on demand). 

Kurkijarvi et al. were the first to demonstrate the feasibility of seg-mented-flow bioluminescence assays by use of a bioreactor packed with bacterial bioluminescent enzymes immobilized on Sepharose 4B [60]. The packed glass colunrn used was placed in front of the photomultiplier tube of a luminometer. The luminescence signal obtained was linearly related to the NADH concentration from 1 pmol to 10 nmol for sample volumes of 2-20 pL. In the region of 400 NADH assays could be performed with a single enzyme column, with no appreciable change in sensitivity or accuracy. However, problems arising from packing or disruption of the matrix were encountered after 4 days of intensive use. 

GPC supports based on dextran (e.g., Sephadex) are cleaned from residual proteins by rinsing with five to ten bed volumes of 2 M NaCl, 0.1 M NaOH, or detergent as 0.1% Tween 20. Equilibrate the column after cleaning with ten bed volumes of elution buffer. Agarose-based beds, e.g., Sepharose or Biogel A, are freed from pollutions by sodium chloride, detergent, or 6 M urea. 

Ten grams of Sepharose are sucked off on a sintered-glass funnel. First wash with 3 vol. water, then with 3 vol. 30% acetone in water (v/v), and then with 3 vol. 60% acetone (v/v) in water. Suspend the gel in 10 ml of 60% acetone and cool to 0 °C. Add the needed amount CDAP dropwise (cf. Table 3.9) followed by the respective amount of triethylamine (TEA) to the stirred gel suspension. Suck off the gel after 2 min and wash with a tenfold volume of Soln. D. The activated gel is stable for 1 h. 

Fig. 6. Elution profiles of three alkyl 2-acetamido-2-deoxy- -D-glucopyranosides 4a (a. n=8 b. n=ll c. n=14) in gel filtration chromatography on sepharose 4B (exclusion 2.10 Da, exclusion volume 10 mL) monitored by and radioactivity countings (normed to 1.0 for reason of convenience). ( ) H countings of H-labelled phosphatidyl choline (x) C...

Pharmacia s Q- and S-Sepharose anion- and cation-exchange resins For the anion-exchange process it was found that two step changes, simultaneous in pH and salt concentration were necessary to carry out the anion-exchange separation. A 0.01 M sodium acetate buffer, pH 5.8, was used forthe starting state orfeed loading buffer. After the whey feed was loaded onto the column, one column volume of this... 

Stipe powder of C. comatus (100 g) was extracted three times with 1 L 95% ethanol under reflux for 2 h to remove lipid, and the residue was extracted three times with 2 L distilled water for 2 h at 80 °C with intermediate centrifugation (2000 x g, 15 min). After concentrating the collected aqueous supernatants to 400 mL (reduced pressure at 40 °C), a precipitation was performed with 3 volumes of 95% ethanol. The precipitate was washed with ethanol and acetone, and then dried at 40 C, yielding crude polysaccharide material. Crude polysaccharide material was dissolved in 100 mL 0.2 M sodium phosphate buffer (pH 6.0), and after centrifugation the solution was applied to a DEAE-Sepharose CL-6B column. 

Wash 10 mL (settled volume) of Sepharose 4B with 1 L of water by vacuum filtration. Resuspend m 18 mL of water (do not allow the Sepharose to dry out)... 

Wash 100 mL Sepharose 4B (settled volume) with 1 L of water by vacuum filtration. 

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