Systematic comparisons of various spectrophotometric and colorimetric methods to measure concentrations of protein, peptide and amino acid: detectable limits, linear dynamic ranges, interferences, practicality and unit costs.

There is limited and inconclusive information regarding detectable limits and linear dynamic ranges of various quantitative protein assays. We thus performed systematic comparisons of seven commonly used methods, including direct spectrophotometric quantitation at λ205 and λ280 nm (A205 and A280, respectively), bicinchoninic acid (BCA), Biuret, Bradford, Lowry and Ninhydrin methods. Purified BSA, porcine kidney extract, tryptic digested peptides derived from purified BSA, and glycine, were used as representative purified protein, complex protein mixture, peptide and amino acid, respectively. Bradford method was the most sensitive assay (LOD=0.006 mg/ml) and had the widest range of detectability (LOD-UOD=0.006-100mg/ml) for purified protein and complex protein mixture. For peptide, A205, A280, Lowry and Ninhydrin methods had a comparable LOD (0.006 mg/ml), but Ninhydrin method had the widest detectability range (LOD-UOD=0.006-100mg/ml). For amino acid, A205 and Ninhydrin methods had a comparable LOD (0.006 mg/ml), but A205 had a wider detectability range (LOD-UOD=0.006-6.250 mg/ml). Biuret method offered the widest linear dynamic range for purified protein and complex protein mixture (0.391-100mg/ml), A280 offered the widest linear dynamic range for peptide (0.024-6.250 mg/ml), and Ninhydrin method offered the widest linear dynamic range for amino acid (0.024-0.195 mg/ml). Both Laemmli's and 2-D lysis buffers had dramatic interfering effects on all assays. Concerning the practicality and unit costs, A205 and A280 were the most favorable. Among the colorimetric methods, Bradford method consumed the least amount of samples and shortest analytical time with the lowest unit cost. These are the most extensive comparative data of commonly used quantitative protein assays that will be useful for selecting the most suitable method for each study.

Penaeus monodon SERPIN, PmSERPIN6, is implicated in the shrimp innate immunity.

Serine proteinase inhibitors (SERPINs or serpins) have been found in a diverse range of organisms. Herein, eight serpin genes, namely PmSERPIN1 - 8, were identified from the Penaeus monodon EST database (http://pmonodon.biotec.or.th/home.jsp). Among those, PmSERPIN6 was selected for further characterization. Tissue distribution analysis revealed that PmSERPIN6 transcripts were expressed in the lymphoid organ, hemocyte, heart and gill, but not in the hepatopancreas. Semi-quantitative RT-PCR analysis at 0-48 h after pathogen challenge demonstrated that the PmSERPIN6 gene transcript expression levels in hemocytes was slightly decreased after systemic white spot syndrome virus (WSSV) injection but remained unchanged upon Vibrio harveyi injection. Interestingly, immunocytochemistry using anti-PmSERPIN6 polyclonal antiserum showed an increase in the number of PmSERPIN6 producing hemocytes at 72 h after both WSSV and V. harveyi injections indicating that the expression of PmSERPIN6 responded to pathogen in the late phase of infection. Our results suggest a likely important function of PmSERPIN6 in the shrimp's defense against invading pathogens.