Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.Download video file.^{(121M, mp4)}     

Somatic Embryogenesis and Plantlet Regeneration of Melia azedarach L (Ghora Neem) from Cotyledonary Segments

Embryogenic calli were obtained within 3–4 weeks on MS medium supplemented with 3% sucrose, 200 mg I-1 caseinhydrolysate, and 3 mg l^?1 each of 2,4-D and NAA from 3–4 days imbibed seeds. Heart-shaped embryos differentiated when growth regulators were withdrawn gradually. Quantification of somatic embryo formation showed a direct relationship between initial response, somatic embryo formation and its subsequent germination. The somatic embryos germinated into emblings with 90% conversion frequency on MS medium containing 2% sucrose.     

Novel expression system for Corynebacterium acetoacidophilum and Escherichia coli based on the T7 RNA polymerase-dependent promoter

The industrially important species of corynebacteria viz. Corynebacterium acetoacidophilum appear to be alternative hosts for recombinant protein production; despite many efforts, a strong promoter-based system in corynebacteria has not been established so far. Described here is a T7 promoter-based expression system which was functional in both gram-positive C. acetoacidophilum and gram-negative Escherichia coli in an external inducer independent manner. This is the very first report of a T7 expression system for Corynebacterium sp. Also, it is a useful addition in the existing T7 expression systems of E. coli.     

Evolution of tryptophan biosynthetic pathway in microbial genomes: a comparative genetic study

Biosynthetic pathway evolution needs to consider the evolution of a group of genes that code for enzymes catalysing the multiple chemical reaction steps leading to the final end product. Tryptophan biosynthetic pathway has five chemical reaction steps that are highly conserved in diverse microbial genomes, though the genes of the pathway enzymes show considerable variations in arrangements, operon structure (gene fusion and splitting) and regulation. We use a combined bioinformatic and statistical analyses approach to address the question if the pathway genes from different microbial genomes, belonging to a wide range of groups, show similar evolutionary relationships within and between them. Our analyses involved detailed study of gene organization (fusion/splitting events), base composition, relative synonymous codon usage pattern of the genes, gene expressivity, amino acid usage, etc. to assess inter- and intra-genic variations, between and within the pathway genes, in diverse group of microorganisms. We describe these genetic and genomic variations in the tryptophan pathway genes in different microorganisms to show the similarities across organisms, and compare the same genes across different organisms to find the possible variability arising possibly due to horizontal gene transfers. Such studies form the basis for moving from single gene evolution to pathway evolutionary studies that are important steps towards understanding the systems biology of intracellular pathways.     

Self-diffusion of zinc and iron in soils as affected by pH,CaCO3, moisture,carrier and phosphorus levels

Summary Self-diffusion coefficients of zinc and iron were determined in acid soil of Palampur and alluvial soil of Ludhiana under varying pH, CaCO₃, moisture, carrier and phosphorus levels. Increase in pH caused tremendous reductions in self-diffusion coefficients (D_a) of both zinc and iron in soil. The selfdiffusion coefficients of both these elements were drastically reduced as a result of CaCO₃ application. The D_a values of zinc and iron increased with the decrease in moisture tension and increase in carrier and phosphorus levels. The decrease in D_a values were associated with increase in capacity factor.