One-dimensional and two-dimensional polyacrylamide gel electrophoresis: a tool for protein characterisation in aquatic samples

Dissolved organic nitrogen (DON) represents the least understood part of the nitrogen cycle. Due to recent methodological developments, proteins now represent a potentially characterisable fraction of DON at the macromolecular level. We have applied polyacrylamide gel electrophoresis to characterise proteins in samples from a range of aquatic environments in the Everglades National Park, Florida, USA. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that each sample has a complex and characteristic protein distribution. Some proteins appeared to be common to more than one site, and these might derive from dominant higher plant vegetation. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) provided better resolution; however, strong background hindered interpretation. Our results suggest that the two techniques can be used in parallel as a tool for protein characterisation: SDS-PAGE to provide a sample-specific fingerprint and 2D-PAGE to focus on the characterisation of individual protein molecules.

Expression of Bf/C2 Gene(S) in the Japanese Medaka Fish

Complement factor B and C2 are two central serine proteases of the alternative and classical complement pathways, respectively, that serve as the catalytic subunits of the C3 convertase. Research has been completed using a female Japanese medaka fish, (Oryzias latipes), and other teleost and elasmobrach species in order to isolate eDNA clones and perform linkage analysis of the Bf/C2 gene(s). To further analyze the evolution of the complement system in teleosts, different tissues than the ones from previous studies of medaka fish were analyzed for the constitutive gene expression of factor B and C2. Bf/C2 sequences were amplified by reverse transcription-polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Agarose gel electrophoresis was used to visualize sample bands and to calculate the concentration of gene expression of the Bf/C2 gene(s) in each tissue. All five tissue types, kidney, liver, muscle, testis, and spleen from a male medaka fish demonstrated Bf/C2 gene(s) expression, confirming that the messages of Bf/C2 gene(s) are distributed throughout the medaka fish. Tissues of the spleen, liver, and kidney contained the highest concentrations of expression of Bf/C2 gene( s ), while tissues of the muscle and testis contained the lowest concentrations. This research also determined that RT-PCR allowed for more sensitive analysis of gene expression than other molecular biology techniques such as Northern blotting analysis.