Gradient mixer

Give 8.1 ml of the acidic solution (mixture as given in Table 2.10 without Soln. C) into the ds chamber of the gradient mixer, open the connecting valve for a moment to let air out, then pour 8.1 ml of the basic solution (mixture as given in Table 2.10 without Soln. C) into the Is chamber. Add Soln. C to start polymerization, switch on the magnetic stirrer, and open both the valves. 

Prepare a linear gradient from Soln. B and Soln. C in a centrifuge tube (e.g., 13 ml Ultra-Clear for Beckman SW 41 rotor 6 ml of Soln. B and Soln. C each) at the day before run. If a gradient mixer is used, e.g., according to Fig. 2.2, place tbe centrifuge tube either in a slanting position, start with Soln. C, and let flow along the tube... 

Prepare solutions of 40%, 30%, and 10% sucrose (w/v) in sucrose gradient buffer. Place 500 pL of 40% sucrose solution in a 13.2-mL ultracentrifuge tube. Layer a 10%-30% sucrose gradient on top of the 40% cushion, using a gradient mixer. 

To achieve a faster separation it is also possible to start the gradient earlier to compensate the dead volume between the gradient mixer and the trap column. On our instrument we have a dead volume of about 1.3 (xL. At a flow rate of 300 nL/min we start the gradient 4 min before the valve switches. 

Behnke and Bayer published a similar approach for gradient elution PEC [51]. The schematic view is shown in Fig. 2.17. A gradient mixer and a HPLC pump were combined with a modular CE system. A post-injection splitter was used and sample introduced by a conventional HPLC six-port injector. A grounded stainless-steel T-piece was used to split both eluent and sample. The electrolyte reservoir on the inlet side of the separation capillary was connected to the splitter by a homemade interface. 

Any HPLC or FPLC machine containing a pump, gradient mixer, UV detector, HPLC columns, and fraction collector can be used. UV detection should be done at 215 nm. Use solvents that do not show absorbance at 215 nm (i.e., acetonitrile, water, and trifluoroacetic acid [TFA]) (see Note 10). For MicroHPLC we use a Smart HPLC system (Pharmacia). 

Figure 4-25. Preparation of gradient gels. Gradient gels are formed using a miniaturised version of the gradient mixer described for use in chromatography. The two vessels are filled with the acrylamide/bis-acrylamide mixtures,...

Fignre 4-8. Gradient mixer. (Courtesy of Pharmacia Fine Chemicals. Inc.)... 

Figure 4-13. Assembly of an ion exchange column, gradient mixer, and magnetic stirring motor. Arrows indicate points where the column is closed with pinch clamps.

Deaerate after first four solutions are mixed and add rest (persulfate last). Prepare the linear gradient with a gradient mixer equipped with chambers of equal cross-sections. The top of the gradient should be at least I cm below the gel strip and immediately overlayered with 1 ml of a 0.1 % SDS solution. 

Small-scale protein purification protocol is suitable when seminal fluid or prostate tissue is used as starting material. Perform all the steps for small-scale protein purification in +4 °C (or cold room). Same buffers than in mass-scale purification are used. Prepare your own columns using strong anion exchanger matrix such as QAE Sephadex A-25, and for gel filtration matrix use e.g. Sephacryl S-200 HR. L-(+)-tartrate afiSnity column is prepared in the same way than for mass-scale purification method. Use peristaltic pumps, gradient mixer and fraction collector in +4 °C. 

In the following order Start the magnetic stirrer, open the gradient mixer outlet, open the valve between the chambers, and start the peristaltic pump. Fill the tubes and take care to stop the pump before air is introduced into the bottom of the tube. Withdraw the capillary carefully. 

Gradient mixer, magnetic stirrer and pump Ultracentrifuge (Beckman)... 

Ultracentrifuge, SW55, 5ml tubes Gradient mixer, magnetic stirrer and pump Scintillation-counter, vials and -liquid Rotating wheel (or rocking table)... 

In the case of gradient elution the low flow rates required a considerable reduction of the retardation volume between static gradient mixer and column head and photodiode... 

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