56 



was allowed to stand for 10 min followed by centrifugation at 5,000g (10°C) 

 for 25 min. The pellet was dissolved in 2.5 volumes (v/v) phosphate 

 buffer (pH 7.6) and the suspension was made up with PEG to 12% (v/v). 

 Again, the suspension was allowed to stand for 10 min and then centrifuged 

 at 5,000g (10°C) for 25 min. The pellet was resuspended in 1/4 volume 

 phosphate buffer and cooled to 0°C before adding an equivalent volume of 

 50% ethanol (-20°C). Following centrifugation at lO.OOOg (4°C) for 25 min, 

 the precipitate was dissolved in 1/4 volume phosphate buffer and the 

 suspension was dialyzed overnight (4°C) against 4L 0.1 M sodium phosphate 

 buffer (pH 7.6). After dialysis, the antibody preparation was made up 

 with NaNj to 0.1% and stored in the refrigerator until needed. 



Molecular Weight Determination of Anti -lobster PPO Antibody 



Protein content of the antibody preparation was also quantitated 

 using the Bio-Rad Protein Assay kit. The molecular weight of the antibody 

 preparation was determined using SDS-PAGE (reduction condition) according 

 to the method of Laemmli (1970). Mini slab gels (7 cm x 8 cm) at 1.0 mm 

 thickness, consisting of stacking gel (4% acrylamide/ 0.1% bisacryl amide) 

 and separating gel (7.5% acrylamide/ 0.2% bisacryl amide) were prepared 

 according to the Mini-Protein II Dual Slab Cell Instruction Manual (Bio- 

 Rad, 1985b). Antibody preparations were diluted with 4 volumes of SDS 

 sample buffer containing ^-mercaptoethanol , and heated for 4 min at 95°C. 

 After 30 /xg aliquots were applied to each sample well and electrophoresis 

 was carried out for 35 min at a constant voltages of 200 V. An SDS-6H 

 high Molecular Weight Protein Standards kit (Sigma) containing carbonic 

 anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000), 



