Rehydratable gels

Immobiline gels are cast in slaTndard vertical cassettes on GelBond PAG supports. After polymerization the gel must be washed to remove unreacted catalysts and unpolymerized Immobilines. The gel swells during this process and must be dried back to its original weight. It is, therefore, much preferable to use a rehydratable gel system, where after washing the gel can be dried, stored and rehydrated for use (see above). 

Since rehydratable gels are polymerized in the presence of a defined buffer, any effects of composition, pH range, batch variation or storage-induced changes of the carrier ampholytes are eliminated. The problems Involved in achieving reproducible polymerization of... 

Table III. Electrical Conditions on Isoelectric Focusing in Rehydratable Gels (60-120 m).

Figure 3. The effect of separators on ultrathin-layer isoelectric focusing in rehydratable gels. Gels (120 jjim) containing 10% Dextran 35 plus 3% glycerol were rehydrated with 3% Servalyt pH 4-6 plus 5%, glycerol with the indicated concentration of different separators. On the left, a mixture of four marker proteins MYH-horse myoglobin, CAR-carbonic anhydrase, LAC-(3-lactoglobulin,...

Rehydratable gels were obtained from Micro-Map, Inc., Boca Rotan, FL, at both 3 per cent T, 3.5 per cent C and 5 per cent, T 3.5 per cent C. (T refers to the per cent of monomer present, while C is defined as the per cent of cross-linker relative to T. The per cent of the gel is the total of these two values) For comparative studies conventional gels were prepared at the same acrylamide concentration from the same bottle of two times recrystallized acrylamide (Serva). Acrylamide was recrystallized also from chloroform as previously described [2]. These gels were 250 or 375 1 thick and were bonded to silanized glass plates, or covalently bonded to GelBond Pag 7 mil mylar sheets (Marine Colloids, Rockland ME ) using previously published methods 118-191 and were randomized between the two laboratories. Single lots of ampholytes from various manufacturers were used for the comparative studies in each laboratory to eliminate lot variation. 

Table III. Run parameters for a rehydratable gel separated on an MRA Cold Focus apparatus. Gel, 3 % T,3.5 % C, 250 l thick, ambient temperature 23° C, Ampholyte 4 per cent Servalyte pH 3-7 in 10 per cent aqueous glycerol with a distance of 7 cm between electrode wick edges. Catholyte, 1.0 M NaOH, anolyte 1.0 M H3PO4. Time given in minutes.

Figure 3. A) Hemoglobin standards A, F, S and C, AS and C separated on a five per cent T, pH 6-8 gradient of Pharmalyte. B) Same samples on a similar rehydratable gel. Both A and B were made with the same base reagents at the same time and were bonded to silanized glass plates.

Figure 6. A) Separation of cell extracts 113 and 160 on a three per cent T rehydratable gel with 4 per cent pH 3-10 Servalyte and 7 cm between wick edges. B) Same samples separated on a five per cent T gel.

Figure 7. Comparison of densitometric results on cell extract 160 separations on randomized seven month old rehydratable gels in two different laboratories. The NBS separation was scanned on an LKB laser densitometer and MUSC gel was scanned on a Biomed SL-2D soft laser densitometer.

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