Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution.

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins.

Mechanism of reversible fluorescent staining of protein with epicocconone.

[reaction: see text] Epicocconone is the active ingredient in Deep Purple Total Protein Stain and responsible for the apparent noncovalent staining of proteins in polyacrylamide gel and electroblots. Reaction of epicocconone with amines has shown that epicocconone reacts reversibly with primary amines to produce a highly fluorescent enamine that is readily hydrolyzed by base or strong acid such as in conditions used in post-electrophoretic analysis such as peptide mass fingerprinting or Edman degradation.

A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis.

Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm.

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line.

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 microM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching.