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.

The expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by glucocorticoids requires a glucocorticoid response unit (GRU), which consists of two non-conserved glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three cofactor elements (gAF1-3). DNA-binding accessory proteins are often essential for gene regulation, and the products of these genes play important roles in metabolism, development, and various defense responses, yet their necessity is poorly understood. Previously, there have been no reports on the quantitative, real-time homogeneous analysis of protein-DNA co-existing interactions in complex media, such as nuclear extracts. In this paper, we investigated protein-DNA interactions in nuclear extracts using quantitative, real-time equilibrium, and stop-flow fluorescence anisotropy measurements. The results showed that GR binds weakly to the GR1-GR2 elements compared to palindromic sequences or conserved glucocorticoid response elements (GREs). However, the presence of either gAF1 or gAF2 elements within GR1-GR2 can create a high-affinity binding environment for GR. At nanomolar concentrations, GR can bind and dissociate with palindromic GRE in multiple rounds within 100 milliseconds. The gAF1 or gAF2 element binds to two different cofactors, COUP-TF/HNF4 and HNF3, respectively, thereby differentially slowing down the dissociation rate of GR. [1] Immunoplasmic probes containing fluorescent labels and 1.4 nm gold clusters were prepared by covalently coupling the nanogold cluster label to the fluorescent portion of the Fab' antibody fragment. These novel immunoconjugates allow for the collection of two sets of complementary data from a single labeling experiment, one from fluorescence microscopy and the other from electron microscopy. Due to the use of the Fab' fragment, the entire probe is smaller than the complete IgG molecule. The fluorescence properties of the novel probes were investigated using a simple fluorescence assay. These probes were used to locate the precursor mRNA splicing factor SC35 in the nucleus of HeLa cells by fluorescence microscopy and electron microscopy, and to label leukocyte microtubules; labeling imaging was performed by fluorescence microscopy and observed by various optical methods and electron microscopy after silver enhancement. In addition, combined probes of gold nanoparticles with Texas red, Cy3, rhodamine B and AMCA were prepared. Preliminary experiments showed that these probes had similar properties to those of combined probes of fluorescein and gold clusters. Fluorescent and gold cluster probes further improved the correlation between fluorescence microscopy and electron microscopy, and can also be used to check the labeling of samples before electron microscopy processing. [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.)