SDS-PAGE
MP isolated from red sea bream was dissolved in NaCl solution
(3.5 g/100 g) at the ratio of MP:NaCl solution of (1:9) in order to
obtain the soluble MP. The soluble MP was determined for protein
concentration by Biuret method before mixing with treatment
buffer, containing SDS and b-mercaptoethanol (BME) prior determination
of protein pattern using SDS-PAGE according to Laemmli
(1970). The protein sample (20 mg) was loaded onto the gel made
from 4 g/100 g acrylamide as stacking gel and 10 g/100 g acrylamide
as running gel. The samples were separated using a mini Protean II
unit (Bio-Rad Laboratories Inc., Richmond, Calif., USA). The protein
patterns of SP were also analyzed as above except the running gel
was 12.5 g/100 g acrylamide.
MP mixture (incubated at 4 C for 6 h) was mixed with 10 times
of SDS (5 g/100 g) solution before boiling for 10 min. The solution
was centrifuged at 2000 g for 15 min before centrifuging and
collected the supernatant as the soluble protein extract. Protein
concentration in the supernatant was estimated by the Biuret
method. The soluble protein was mixed with treatment buffer
containing BME in order to break down the disulfide linkages. The
pattern of soluble protein was analyzed by SDS-PAGE.