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.