Proteins fast green

Protein (total) Fast green pH2 Ninhydrin—Alloxan Schiff s Mercuric-bromphenol blue... 

The dye l-Anilino 8-Naphthalene Sulfonic acid (ANS) has high specificity for protein. It fluoresces only when bound to protein [30]. In smears and handsections (i.e. unembedded materials) we have never observed it to effect emulsion stability in the manner more traditional protein dyes such as Coomassie Brilliant Blue or Fast Green often do. This relative pH independence probably is due to the mode of action of this dye. It becomes fluorescent in hydrophobic pockets on protein molecules [30] in contrast to the ionic bonding necessary for Fast Green FCF and Coommassie Blue [22]. We have not observed a strong cross-reaction with lipids, either, although a fluorescence of different spectral characteristics sometimes is seen. 

For sections of embedded material, we generally do not use ANS to localize protein. Instead we use Coomassie Brilliant Blue or Fast green FCF which are used as diachromes. Either dye is used at 0.1%W/V in 7% acetic acid [22], Slides having a puddle of stain over the sections are gently warmed for up to 5 minutes, rinsed with distilled water and allowed to dry before mounting in immersion oil for observation. Figure 5 shows an example of cream cheese treated in this manner to reveal protein. 

Figure 5 Cream cheese fixed in glutaraldehyde and embedded in glycolmethacrylate. The section was stained for protein with Fast Green. Protein has variable staining intensity, perhaps reflective of compositional differences.The fat (white) does not stain, (x 1300).

Nitrocellulose-bound proteins can be stained with amido hlack lOB (Towbin et al., 1979), CBB (Burnette, 1981), and several other dyes (aniline black. Ponceau S, fast green, toluidine blue Gershoni and Palade, 1983). The recently developed immunostain for proteins on nitrocellulose filters (Wojtkowiak et al., 1983) is very sensitive (Section 16.3.1). 

Coomassie brilliant blue R-250 (Fazekas de St. Groth, S., Webster, R.G., and Datyner, A., Biochem. Biophys. Acta., 71,377, 1963) requires an acidic medium and then is believed to be electrostatically attracted to the NHj groups of the protein. It is three times more sensitive than Fast green. In a 0.01 M citrate buffer at pH of 3, it has a at 555 nm and at 549 nm for the protein dye complex. 

Acid fast green staining of proteins of polytene chromosomes Protocol 24... 

Chattoraj M, King BA, Bublitz GU, Boxer SG (1996) Ultra-fast excited state dynamics in green fluorescent protein multiple states and proton transfer. Proc Natl Acad Sci USA 93 8362-8367... 

Kummer AD, Kompa C, Lossau H, Pollinger-Dammer F, Michel-Beyerle ME, Silva CM, Bylina EJ, Coleman WJ, Yang MM, Youvan DC (1998) Dramatic reduction in fluorescence quantum yield in mutants of green fluorescent protein due to fast internal conversion. Chem Phys 237 183-193... 

A bright cyan-green fluorescent protein was isolated from Clavu-laria coral [86]. Since one of the intermediates displayed fast bleaching, a screen for more photostable variants was performed. The optimized monomeric variant was named teal fluorescent protein 1 (mTFPl). It has an excitation and emission maximum at 462 and 492 nm, respectively, so this protein is spectrally located in between CFP and GFP. With an extinction coefficient of 64,000 M 1 cm-1 and a quantum yield of 0.85 mTFPl is a very bright fluorescent protein. 

Thompson, P.R. and Fast, W. (2006) Histone citrullination by protein arginine deiminase is arginine methylation a green light or a roadblock ACS Chemical Biology, 1, 433-441. 

Ultra fast dynamics in the excited state of wild-type Green Fluorescent Protein... 

The most frequently used protein assay is based on a method after Bradford (Bradford, 1976), which combines a fast and easily performed procedure with reliable results. However, the Bradford assay has sensitivity limitations and its accuracy depends on comparison of the protein to be analyzed with a standard curve using a protein of known concentration, commonly bovine serum albumin (BSA). Many commercially available protein assays such as those from Pierce or BioRad rely on the Bradford method. The assay is based on the immediate absorbance shift from 465 nm (brownish-green) to 595 nm (blue) that occurs when the dye Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. Coomassie dye-based assays are known for their non-linear response over a wide range of protein concentrations, requiring comparison with a standard. The dye is assumed to bind to protein via an electrostatic attraction of the dye s sulfonic groups, principally to arginine, histidine, and lysine residues. It also binds weakly to the aromatic amino acids, tyrosine, tryptophan, and phenylalanine via van der Waals forces and hydrophobic interactions. 

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