Shrimp 2D protein electrophoresis

Description

2-DE proteomics allows greatly improved resolution for differential display proteomics (comparison proteomics). It also provide improved support for studies on eg. posttranslational modification and the selection of protein of interest for subsequent amino acid sequencing.

Brief protocol

Protein preparation

The frozen stomachs from three shrimp were ground to a fine powder at -80℃. The powder was then suspended in a three-fold dilution of PBS buffer containing protease inhibitor cocktail (applied according to the manufacturer’s protocol, Roche Diagnostics, Mannheim, Germany). After centrifugation at 3000g (30 min, 4℃), the supernatant was collected and a TCA/DTT mixture was added (final concentration: 10% w/v TCA and0.1% DTT). After standing on ice for 30 min and another centrifugation (10,000g, 30 min, 4℃), the supernatant was discarded and the pellet resuspended in acetone containing 0.1% DTT. The sample was spun again (10,000g, 30 min, 4℃),and the pellet-dried under vacuum and then solubilized in rehydration buffer (9.8 M urea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer [pH 4–7 or 3–10; Amersham Biosciences]). After final centrifugation (10,000g, 30 min, 15℃), the supernatant, which contained the soluble protein fraction, was used as a 2-DE sample. Protein concentration of 2-DE (two-dimensional electrophoresis) samples was standardized using a 2-D Quant Kit (Amersham Biosciences). The protein sample was incubated at -80℃ until completely electrophoresis separation on 2-DE analysis.

Protein separation

The first dimension of the 2-DE, isoelectricfocusing (IEF), was performed in 13 cm Immobiline DryStrip gel (Amersham Biosciences) using an integrated system, the Ettan IPGphor (Amersham Biosciences), where rehydration with the sample and IEF are performed automatically. Two pH gradient strips were used: linear pH 4–7 and 3–10. Each sample (250 mg protein) was dissolved in 250 ml rehydration buffer with a trace of bromophenol blue and placed in the base well of an IPGphor stripholder. An IPG strip was then placed on the top of the sample, and after rehydration in the IPGphor (16 h at 50 V), automatic IEF was performed using the following step voltage focusing protocol: 1 h at 300 V, 1 h at 500 V, 2 h at 1000 V, 2 h at 4000V, 10 h at 8000 V and 24 h at 50V. All the above procedures were carried out at 20℃. After the first dimensional IEF, the IPG strips were equilibrated in a sodium dodecyl sulfate (SDS) equilibration buffer (6M urea, 2% SDS, 30% glycerol, 50mM Tris–HCl, pH 8.8) containing 1% DTT for 15 min. The IPG gel strips were then removed to another equilibration buffer containing 2.5% iodoacetamide and equilibrated for a further 15 min. The equilibrated IPG strips were then placed onto a polyacrylamide gel that consisted of 14% acrylamide, pH 8.8, for the separating gel, and 4% acrylamide, pH 6.8, for the stacking gel. The second dimensional separation was run at 20 mA per gel at 15℃ for 5–6 h. At the end of each run, the gels were stained with Sypro Ruby, and the protein patterns of the gels were scanned using a Typhoon 9400 scanner (Amersham Biosciences). Gel image matching was done using PDQuest software (Bio-Rad).

References

1. H.C. Wang, H.C. Wang, J.H. Leu, G.H. Kou, Andrew H.-J. Wang and C.F. Lo. (2007) Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection. Developmental & Comparative Immunology 31: 672-686.

Some of our publications that have used this platform

1. H.C. Wang, H.C. Wang, J.H. Leu, G.H. Kou, Andrew H.-J. Wang and C.F. Lo. (2007) Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection. Developmental & Comparative Immunology 31: 672-686.
2. Wang HC, Wang HC, Kou GH, Lo CF, Huang WP. (2007). Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV). Dis. Aquat. Org. 74:179-189.