Protein autofluorescence

Wolff M, Kredel S, Wiedenmann J, Nienhaus GU, Heilker R (2008) Cell-based assays in practice cell markers from autofluorescent proteins of the GFP-family. Comb Chem High Throughput Screen 11 602-609... 

However, engineering of fluorescent marker proteins to determine subcellular protein localizations and associations in planta can be quite challenging since plant cells contain a number of autofluorescent compounds (e.g., lignin, chlorophyll, phenols, etc.,) whose emission spectra interfere with that of the most commonly used green or red fluorophores and their spectral variants... 

Billinton N, Knight AW. Seeing the wood through the trees a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal. Biochem. 2001 291 175-197. 

The neuronal ceroid lipofuscinoses (CLN), also referred to as Batten s disease, are a group of disorders characterized by the accumulation of autofluorescent lipopigments. Clinical hallmarks include blindness, seizures, cognitive and motor decline and early death. Age of onset varies from infancy to adulthood. Eight genetic forms have been identified [4]. Two involve lysosomal acid hydrolases. CLN1 codes for palmitoyl protein thioesterase 1. Clinically it presents most often in infancy and leads to loss of active movement and visual contact by 3 years of age. It is most common in Finland, where its incidence is 1 20,000. CLN2 codes for a lysosomal pepstatin-insensitive acid protease. 

Fluorescence occurs when radiant energy is absorbed and then, almost instantly, some of the energy is re-emitted, usually at a longer wavelength. Primary fluorescence (autofluorescence) occurs in flavo-proteins (13), plant cell wall materials such as lignin (7), and in flagella (14). Secondary fluorescence is when a material binds a fluorescent dye... 

GFP is an autofluorescent protein which has become a powerful tool in molecular and cell biology.[14,15] The GFP chromophore is formed autocatalytically from cyclization and oxidation of a Ser-Tyr-Gly tripeptide (in wild type A. Victoria) resulting in the p-hydroxybenzylidene-imidazolinone (HBI) molecule shown in Sketch 1. The R and R groups represent the covalent... 

Autofluorescence can be very useful for localizing proteins without staining or other perturbations of the product but it can be a major issue in the interpretation of stained dairy foods. It remains as background fluorescence in the presence of fluorochromes giving similar colors, particularly the protein dye, ANS and FTTC-conjugated molecules such as lectins. 

Proteomic Fluorescence microscopy In situ visualisation of cellular/ECM protein with fluorescence-labelled antibody Multiplexing capability (typically 3 fluorophores). Extra- and intracellular antigen location on opaque biomaterials. Issues with bleaching and autofluorescence. Yes/no... 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

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