Establishment of two-dimensional electrophoresis( 2-DE) technique in low abundance hemolymph proteome of Penaeus monodon

We optimized the two-dimensional electmphoresis technique for analyzing hemolymph in Penaeus monodon. To obtain high resolution 2-DE patterns, we examined the protein extraction, IPG-strip length, separation scope of IPG strip, sample loading amount and focusing conditionby PDQuest software. Results indicate that PEG 6000removed high abundance protein in haemolymph faster and more effectively, and increased the concentration of low abundance protein. The protein preparation with 80% TCA-acetone extraction treatment in isoelectric focusingwas effective. The optimum 2-DE patterns in the proteins of Penaeus monodonwere successfully separated by using pH 3~10 NL IPG strips with 18 cm length and 300 g protein sample. The study provides stable and replicable method for relevant studies on hemolymph proteame of P.monodon and other similar crustaceans.