Fluorescent dye

[1]. Accessory factors facilitate the binding of glucocorticoid receptor to the phosphoenolpyruvate carboxykinase gene promoter. J Biol Chem. 2001 Oct 26;276(43):39885-91.

[2]. Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy. Microsc Res Tech. 1998 Jul 1;42(1):2-12.

Glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene requires a glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three accessory factor elements (gAF1-3). DNA-binding accessory proteins are commonly required for the regulation of genes whose products play an important role in metabolism, development, and a variety of defense responses, but little is known about why they are necessary. Quantitative, real time homogenous assays of cooperative protein-DNA interactions in complex media (e.g. nuclear extracts) have not previously been reported. Here we perform quantitative, real time equilibrium and stopped-flow fluorescence anisotropy measurements of protein-DNA interactions in nuclear extracts to demonstrate that GR binds to the GR1-GR2 elements poorly as compared with a palindromic or consensus glucocorticoid response element (GRE). Inclusion of either the gAF1 or gAF2 element with GR1-GR2, however, creates a high affinity binding environment for GR. GR can undergo multiple rounds of binding and dissociation to the palindromic GRE in less than 100 ms at nanomolar concentrations. The dissociation rate of GR is differentially slowed by the gAF1 or gAF2 elements that bind two functionally distinct accessory factors, COUP-TF/HNF4 and HNF3, respectively. [1]

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Immunoprobes which incorporate both a fluorescent label and a 1.4 nm gold cluster compound were prepared by covalent conjugation to Fab' antibody fragments of the Nanogold cluster label followed by a fluorescent moiety. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. By using Fab' fragments, the entire probe is smaller than a whole IgG molecule. A simple fluorescence assay was used to investigate the fluorescence properties of the new probes. They were used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy, and also for labeling leukocyte microtubules; labeling was imaged using fluorescence microscopy and, after silver enhancement, by a variety of optical methods and by electron microscopy. Combined Nanogold and Texas Red, Cy3, Lissamine Rhodamine B, and AMCA probes were also prepared, and in preliminary experiments show similar properties to the combined fluorescein and gold cluster probes. The fluorescent and gold cluster probes enable a new degree of correlation between fluorescence and electron microscopy, and may also be used to check labeling of specimens before processing for electron microscopy.[2]

C25H15NO9

473.39

946.14936

117548-22-8

92044394

Yellow to orange solid powder

224ºC

3.429

4

18

10

70

2140

0

C1(=C2C=CC(=O)C=C2OC2C=C(O)C=CC1=2)C1C=CC=CC=1C(=O)ON1C(=O)CCC1=O

QBWKLWIYDVVARL-UHFFFAOYSA-N

InChI=1S/2C25H15NO9/c27-13-2-5-16-19(10-13)34-20-11-14(28)3-6-17(20)23(16)18-9-12(1-4-15(18)24(31)32)25(33)35-26-21(29)7-8-22(26)30;27-13-2-5-16-19(10-13)34-20-11-14(28)3-6-17(20)23(16)15-4-1-12(9-18(15)24(31)32)25(33)35-26-21(29)7-8-22(26)30/h2*1-6,9-11,27H,7-8H2,(H,31,32)

4-(2,5-dioxopyrrolidin-1-yl)oxycarbonyl-2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic acid;5-(2,5-dioxopyrrolidin-1-yl)oxycarbonyl-2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic acid

5(6)-FAM SE; 5(6)-Carboxyfluorescein succinimidyl ester mixed isomers; 4-(2,5-Dioxopyrrolidin-1-yl)oxycarbonyl-2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic acid;5-(2,5-dioxopyrrolidin-1-yl)oxycarbonyl-2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~176.03 mM)

Solubility in Formulation 1: 6.25 mg/mL (13.20 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (4.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)