Elution buffer

TABLE 4.3 Resolution Factors (Rs) of Proteins with Various Ionic Species Added to the Elution Buffer" ... 

TSK-GEL SW columns allow use of elution buffers comprised completely of water-soluble organic solvents, whereas the TSK-GEL PW packings limit organic cosolvent use to a maximum of 50%. 

Stalcup aiid co-workers [14] adapted this method to a continuous elution mini-prep electrophoresis apparatus shown in Fig. 11-3. In this apparatus, the end of the electrophoretic gel is continuously washed with elution buffer. The eluent can then be monitored using an HPLC detector (Fig. 11-4) and sent to a fraction collector where the purified enantiomers, as well as the chiral additive, may be recovered. In this system, the gel configuration was approximately 100 mm x 7 mm, and was aircooled. The number of theoretical plates obtained for 0.5 mg of piperoxan with this gel was approximately 200. A larger, water-cooled gel was able to handle 15 mg of... 

Fig. 17.5. Experimental configuration for integrated bead milling and fluidised bed adsorption 1. Feedstock 2. peristaltic pump 3. bead mill 4. flow through/waste 5. fluidised bed contactor 6. elution buffer 7. fraction collector/ waste 8. loading buffer.

The results are summarised in Table 1. As expected, for the IE samples higher amounts of uronic acid are found with increasing ionic strength of the elution buffer. For the GF samples larger molecules (GFl) contain half as much NS compared to smaller molecules (GF3). 

Elution buffer identical to wash buffer, but additionally containing 3 xFLAG peptide (Sigma) to a final concentration of 0.1 mg/ml... 

Pellet resin and resuspend in elution buffer, collect supernatant, aliquot and store at -80°... 

Elution. Incubate the resin for 30 min with rolling in 500 /il Elution Buffer to competitively elute retained proteins. Collect the supernatant after centrifugation at 9300 xg for 5 min at 4° in a microcentrifuge (10,000 rpm in an Eppendorf 5415R). 

Resuspend the resin in 50 /A of elution buffer E (20 mM Tris-HCl [pH 7.5], 100 mM KC1, 5 mM MgCl2, 0.5 mM /J-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 250 mM imidazole, 10% (v/v) glycerol, 1X Complete Protease Inhibitor Mix tablets [Roche]), briefly vortex, and elute the bound proteins by mixing on a rotator at 4° for 20 min. Please note that a concentration of imidazole and the kind of Roche tablets differs from BB buffer. 

Elute the bound conjugate with 0.1 M glycine, 0.15M NaCl, pH 2.8, or another suitable elution buffer. A neutral pH alternative to this buffer is the Gentle Elution Buffer from Thermo Fisher. If acid pH conditions are used, immediately neutralize the fractions eluting from the column by the addition of 0.5 ml of 1M Tris, pH 8.0, per fraction. 

Glycoproteins generally bind to lectin affinity columns at pH values close to neutrality. Desorption may be achieved in some cases by alteration of the pH of the eluting buffer. The most common method of desorption, however, involves inclusion of free sugar molecules for which the lectin exhibits a high affinity in this elution buffer, i.e. the inclusion of a competing ligand. 

Elovich equation, 1 595 Elution, in ion exchange, 14 410—412 Elution buffer, in affinity chromatography, 6 392... 

Membrane absorbers are continuous chromatographic supports, which circumvent some of the above-mentioned problems of particulate stationary phases. They were originally derived from membrane (filtration) technology. The immobilization of interactive (ionic, hydrophobic, or biospecific) groups on the surface of microfiltration membranes was found to increase the selectivity of certain separation procedure. Ideally such activated membranes, or membrane adsorbers, allow the selective adsorption of certain substances and substance classes, which may subsequently be eluted by means of a stepwise change of the mobile phase (elution buffer). More complete information on the various types of modern membrane technology can be found in some recent reviews [e.g., 31-33]. 

Figure 1 shows a schematic drawing of a CAC apparatus. The apparatus consists of two concentric cylinders standing one inside the other, forming an annulus into which the stationary phase is packed. This annular bed is slowly rotating about its vertical axis. Under isocratic elution conditions the feed mixture to be separated is introduced continuously at the top of the bed at a space that remains fixed in space while the rest of the annulus is flooded with elution buffer. As time progresses, helical component bands develop from the feed point, with... 

The feed is introduced at the top of the annular column at the 0° position. The feed solution is followed by a wash buffer, which is introduced to the annular column through the main inlet port. A 1 vol.% mixture of 2-propanol in a 100 mmol/1 ammonium acetate buffer was used as wash buffer. In the washing zone the nicked DNA followed by the RNA are eluted from the column according to their affinity to the resin. At 180° offset from the feed nozzle the elution buffer (5 vol.%) 2-propanol in 100 mmol/1 ammonium acetate) was pumped to the annulus of the column. The elution buffer was used to strip off the bounded Plasmid DNA. Regeneration of the column was achieved by a 20 vol.% mixture of 2-propanol in 100 mmol/1 ammonium acetate buffer. All of the above-mentioned steps, i. e., feed, wash, elution, and regeneration, were done simultaneously and continuously on the P-CAC system. 

However, it is not only the pH of the eluting buffer that determines the relative elution position of the amino acids, but also the cation concentration of the buffer. Sodium citrate buffer solutions are commonly used and the positive sodium ions compete with the positively charged amino acids for the sul-phonic acid sites on the resin ... 

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