Volume, of gels

Figures 13.8 and 13.9 show the separation of polystyrene standards using a typical mixed-bed column and its calibration plot, respectively. The major advantages of using a large i.d. 10-mm column are low hack pressure and relatively short run times. As seen in Fig. 13.8,10 standards from toluene thru 8.4 X 10 MW can be resolved in a mere 21 min. Because of the large 10-mm i.d. columns, 1.5-ml/min flow rates give a linear velocity equivalent to that of only 0.9 ml/min using a 7.6-mm i.d. column. Also, the gel volume contained in one 10 mm i.d. X 500 mm column is 39.3 ml, whereas a 7.6 mm i.d. X 300 mm column contains only 13.6 ml of gel volume. This bulk volume factor, combined with the large pore volumes of gels, obtains essentially the same resolution as that obtained on three standard 7.6 X 300-mm columns in series, but in about one-half the usual time required using the smaller columns.

Elute the protein with 1.2 x gel filtration column volume of Gel Filtration Buffer (20 mM Tris pH 7.5, 200 mM NaCI, reducing agents as desired) collecting fractions. 

Fig. 4. Phase diagram of gels, fl and v denote the osmotic pressure and the microscopic volume of gel. (v = a3/< >). This Il-v plane corresponds to P-V plane of van der Waals fluids. At % < Xc. n is a convex function of v. At > Xc. a volume phase transition from collapsed to swollen states or vice versa occurs. Note II can be either positive or negative...

Gel-filtration columns may also be packed by using an extension reservoir attached directly to the top of the column Here the total volume of gel and buffer can be poured and allowed to settle without continual topping up. When using an extension reservoir, leave the column to pack until the gel bed just runs dry, and then remove excess gel. 

Each chamber was filled with agarose solution to a depth of 4 mm. After gelation, the agarose samples were exposed to 100-fsw (feet sea water) pressures (i.e., 44.5 psig) for 40 min at 21°C, and then decompressed to atmospheric pressure in accord with one of the seven different decompression schedules tested (see Section 8.1.1). Only bubbles formed in the bottom 3 mm of a given agarose sample were counted, so that the total volume of gel examined in each sample amounted to 0.27 ml. 

The volume of gel extract (12 ml) can be reduced before precipitation by several extractions with n-butanol. One volume of water is taken up by 6 volumes of butanol. A ten-fold concentration does not appear to affect the purity of the DNA recovered after ethanol precipitation. 

The total volume of gel solution will depend on the dimensions of the gel molds used. The dimensions we use (length by width by depth) are 21 X 18 X 0.3 cm, 21 X 18 X 0.6 cm, and 21 X 18 X 0.9 cm. By using deeper molds, we can remove multiple layers from a single gel and assay for enzymes that are separated using the same buffer system and have similar electrophoretic mobilities, thereby conserving materials, time, and space. 

Below are procedures for preparing gels for protein electrophoresis. The volume of gel buffer is for a 21 X 18 X 0.3 cm gel mold unless otherwise noted. 

Add acrylamide and APS to one-half of the total volume of gel buffer and mix well with constant stirring at room temperature. 

Combine the starch and remaining volume of gel buffer in a 500-ml Erlenmeyer side-arm flask stopper the flask and heat (with constant stirring) to 80°. 

The porosity of the hydration product is given by dividing the volume of gel pores per kg of reacted cement by that of the hydration product. This gives x mJ[V + (w/c )/ )J, or 0.30. The value of 0.28 given by Powers and Brownyard was obtained assuming a different value for Kg. It should be noted that this quantity is the volume of gel pores relative to that of all the hydration products, including the CH. 

For proteins, a mathematical model of retention has been developed that works well for Sephadex gels.5 The solute is treated as a sphere of radius rs, while the gel is a network represented by infinitely long, straight rods of radius rx. The rods are randomly distributed, and have an average density of L units of rod length per unit volume of gel. The values of L and rx may be calculated from known dimensions of dextran chains, and then Km may be found from Eq. 14.13 ... 

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