Fluorescent Dye

Alexa 350, Alexa 430, Alexa Fluor 488-azide, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 dyes are a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa names indicate the approximate excitation wavelength maximum in nm). All Alexa dyes and their conjugates are more fluorescent and more photostable than their commonly used spectral analogues listed above. In addition, Alexa dyes are insensitive to pH in the 4-10 range. We evaluated Alexa dyes compared with conventional dyes in applications using various conjugates, including those of goat anti-mouse IgG (GAM), streptavidin, wheat germ agglutinin (WGA), and concanavalin A (ConA). Conjugates of Alexa 546 are at least twofold more fluorescent than Cy3 conjugates. Proteins labeled with the Alexa 568 or Alexa 594 dyes are several-fold brighter than the same proteins labeled with lissamine rhodamine B or Texas Red dyes, respectively. Alexa dye derivatives of phalloidin stain F-actin with high specificity. Hydrazide forms of the Alexa dyes are very bright, formaldehyde-fixable polar tracers. Conjugates of the Alexa 430 (ex 430 nm/em 520 nm) and Alexa 532 (ex 530 nm/em 548 nm) fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges. [1]

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To examine the posttranslational modifications of the C-terminal CCaX motif of bCdc42, we first confirmed palmitoylation of bCdc42 in HEK293 cells. Cells were incubated with the palmitic acid analog 17-octadecynoic acid (17-ODYA) or palmitic acid as a negative control. Protein incorporating 17-ODYA was detected by selective labeling with Alexa Fluor 488-azide via Cu(I)-catalyzed azide-alkyne cycloaddition (click) chemistry. As previously reported, bCdc42 but not Cdc42 was palmitoylated (Fig. 1B). Palmitoylation of bCdc42 was inhibited by 2-bromopalmitate, a nonmetabolizable fatty acid that inhibits palmitoylation (Fig. 1C). Interestingly, a geranylgeranyl transferase inhibitor also blocked the incorporation of palmitate into bCdc42 (Fig. 1C), suggesting that geranylgeranyl modification might be a prerequisite for bCdc42 palmitoylation. To confirm prenylation of bCdc42, cells were incubated with geranylgeranyl-azide or farnesyl-azide. bCdc42 incorporated both prenyl lipid analogs, and incorporation was blocked by prenyltransferase-specific inhibitors (Fig. 1D) [2].

Binding assay. [1]
For the pulldown assay, transfected cells were washed with PBS and lysed with RIPA buffer supplemented with protease inhibitors. Cleared lysates were incubated for 2 h at 4°C with 1 μM GST or GST-RhoGDIα precoupled to glutathione-Sepharose. After three washes with RIPA buffer, the bound proteins were analyzed by immunoblotting. For the in vitro binding assay, 1 μM palmitate- or 17-ODYA-labeled His-bCdc42 was incubated with 1 μM GST or GST-RhoGDIα in binding buffer B (20 mM HEPES-NaOH [pH 7.4], 100 mM NaCl, 3 mM MgCl2, and 1% NP-40) for 1.5 h at 30°C. Samples were further incubated with glutathione-Sepharose for 1 h at 4°C. The resins were collected by centrifugation, and the supernatants were recovered as the glutathione-Sepharose unbound fraction. After three washes with binding buffer B, the bound proteins (bound fraction) were eluted by boiling in binding buffer B containing 1% SDS. SDS was added to the unbound fraction at a final concentration of 1%, and the total volume of each fraction was equalized. The bound and unbound fractions were subjected to click chemistry and immunoblotting.

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Liposome binding assay. [1]
17-ODYA-labeled bCdc42 (1 μM) was incubated with 1 mg/ml of liposomes containing 35% phosphatidylethanolamine, 25% phosphatidylserine, 5% phosphatidylinositol, and 35% cholesterol for 30 min and centrifuged at 16,000 × g for 20 min. The liposome pellets were suspended in binding buffer C (50 mM HEPES-NaOH [pH 7.4], 100 mM NaCl, 3 mM MgCl2) and incubated with GST-RhoGDIα for 30 min. After a final centrifugation, the pellets were suspended in binding buffer C containing 1% SDS. The supernatants were supplemented with SDS (final concentration, 1%), and each sample was used for click chemistry. After click chemistry, samples were precipitated with methanol-chloroform and dissolved in sample buffer. Probe-labeled bCdc42 and total bCdc42 were detected by in-gel fluorescence and CBB stain, respectively.

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Cu(I)-catalyzed azide-alkyne cycloaddition reaction (click chemistry). [1]
Transfected HEK293 cells were cultured in DMEM with 10% FBS for 42 h. Cells were then incubated in DMEM containing 10% dialyzed FBS and 100 μM 17-ODYA or palmitic acid for 6 h. For inhibitor treatment, 5 μM geranylgeranyltransferase inhibitor (GGTI-298) or farnesyltransferase inhibitor (FTI-277) was added to cells 18 h prior to 17-ODYA labeling; 2-bromopalmitate was added to cells 1 h prior to 17-ODYA labeling. To detect prenylation, transfected cells were cultured for 8 h and then incubated in DMEM containing 10% dialyzed FBS and 30 μM geranylgeranyl-azide or farnesyl-azide for 24 h. Cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA buffer (20 mM HEPES-NaOH [pH 7.4], 100 mM NaCl, 3 mM MgCl2, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) supplemented with protease inhibitors (3 μg/ml of leupeptin and 1 mM phenylmethylsulfonyl fluoride [PMSF]). Cleared lysates were immunoprecipitated with anti-FLAG and protein G-Sepharose for 4 h. The immunoprecipitates were washed 3 times with RIPA buffer and suspended in 94 μl of PBS, and 6 μl of freshly premixed click chemistry reagent (final concentrations of 10 μM Alexa Fluor 488-azide or Alexa Fluor 647-alkyne, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 100 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and 1 mM CuSO4) was added. After 1 h at room temperature, the immunoprecipitates were washed twice with PBS containing 1% NP-40 and treated with sample buffer for SDS-PAGE. Probe-labeled proteins were detected by in-gel fluorescence.

Fractionation assay. [1]
Transfected cells were cultured for 8 h and then incubated in the medium containing 30 μM geranylgeranyl-azide and 17-ODYA for 24 h and 6 h, respectively. Cells were harvested with buffer A (20 mM HEPES-NaCl [pH 7.4], 5 mM KCl, and 2 mM EDTA) containing protease inhibitors. Cells were homogenized on ice by 20 passes through a 27-gauge syringe needle. Nuclei and intact cells were removed by centrifugation at 800 × g for 5 min. Postnuclear supernatants were subjected to centrifugation at 100,000 × g for 30 min, and the pellets were suspended in buffer A containing 1% NP-40, 0.5% deoxycholate, and 0.1% SDS. The same detergents were directly added to supernatants. After 1 h of incubation, the samples were subjected to ultracentrifugation and cleared lysates were used for immunoprecipitation, followed by click chemistry.

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Confocal microscopy. [1]
HeLa cells were grown on glass bottom dishes and transiently transfected. At 6 h after transfection, cells were incubated in phenol red-free DMEM with 10% FBS and 25 mM HEPES overnight. Cells were imaged on Zeiss LSM510 META confocal microscope using a Plan-Apochromat 63×/1.4-numerical-aperture (NA) objective lens.

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BRET. [1]
HEK293 cells were seeded on a 12-well plate 24 h prior to transfection. Rluc-bCdc42 wild type or mutants (donor) were transfected alone or with Venus-PTP1b, Venus-giantin, or Venus-K-Ras (acceptor). At 24 h posttransfection, cells were washed with PBS, detached from the plate with PBS containing 2 mM EDTA, and collected by centrifugation at 400 × g for 5 min. Cells were suspended in PBS containing CaCl2 and MgCl2 and transferred to a 96-well plate. Coelenterazine-h (5 μM) was added 10 min before BRET measurement. Luminescence and fluorescence signals were detected using multimode microplate reader Synergy 2 (BioTek). The BRET ratio was calculated by dividing the fluorescence signal (528/20 emission filter) by the luminescence signal (460/40 emission filter). Net BRET was this ratio minus the same ratio measured from cells expressing the donor construct only.

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Luciferase assay. [1]
HEK293 cells were seeded on a 12-well plate and transfected with pEGFP-bCdc42 or mutant bCdc42 plasmids, pSRE-Luc, and pEF-Rluc. At 7 h posttransfection, the medium was replaced with DMEM containing 1% FBS. After 17 h, cells were lysed and subjected to the Dual-Glo luciferase assay system. Expression levels of bCdc42 and its mutants were confirmed and quantified by immunoblotting. Firefly luciferase activity derived from pSRE-Luc was normalized to the Renilla luciferase activity derived from pEF-Rluc. Data were normalized to the expression level of bCdc42 and its mutants.

Stoichiometry of endogenous palmitoylated bCdc42 in mouse neonatal brain. [1]
For the acyl-RAC assay, mouse neonatal brain or adult kidney was homogenized in buffer A containing protease inhibitors with a Dounce homogenizer. Nuclei and intact cells were removed by centrifugation at 800 × g for 7 min. Postnuclear supernatants were subjected to centrifugation at 100,000 × g for 30 min, and the pellets were suspended and incubated in blocking buffer containing 0.1% MMTS at 42°C for 30 min with rotation. Proteins were precipitated by adding 3 volumes of cold acetone. After centrifugation, protein pellets were washed with 70% cold acetone and suspended in binding buffer. An aliquot was saved for the input lane. The remaining sample was used for an acyl-RAC assay as described above. The acyl-RAC samples and input (10% of total) were subjected to immunoblotting with bCdc42-specific and Cdc42 antibodies. The Cdc42 antibody detects both Cdc42 and bCdc42 isoforms. For the RhoGDIα pulldown assay, postnuclear lysates of neonatal brain and adult kidney were subjected to centrifugation at 100,000 × g for 30 min, and the pellets were suspended in binding buffer B. After 1 h of incubation, the samples were subjected to ultracentrifugation, and cleared lysates (2 mg/ml) were incubated for 2 h at 4°C with 0.4 μM GST-RhoGDIα precoupled to glutathione-Sepharose. GST-RhoGDIα unbound and bound fractions were collected. bCdc42 was detected by immunoblotting.

[1]. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem. 1999 Sep;47(9):1179-88.

[2]. Identification of a novel prenyl and palmitoyl modification at the CaaX motif of Cdc42 that regulates RhoGDI binding. Mol Cell Biol. 2013 Apr;33(7):1417-29.

[3]. A review on experimental chemically modified activated carbon to enhance dye and heavy metals adsorption. Cleaner engineering and technology, 2022, 6: 100382.

Effective and low-cost removal of dye and heavy metals from wastewater still is a great challenge for researchers. Adsorption using activated carbon is widely used in removing these toxic pollutants. Physical, chemical, and biological modifications have been studied for improving activated carbon adsorption performance. Literature suggests that chemical modified activated carbon showed maximum adsorption capacity towards dye and heavy from aqueous solution. Chemical modifications, including acid, base, and impregnation, are studied extensively due to reagent availability, easy modification, and tuning facilities of surface functional groups. However, systematic documentation of chemical modifications on activated carbon is required for dye and heavy metals removal efficiency improvement from wastewater. This review focused on the up to date experimental chemically modified activated carbon that showed improved adsorption capacity towards dye and heavy metals from aqueous solution. The available experimental data recommends that an appropriate treatment strategy of a chemical modification process enhanced dye and heavy metals adsorption capacity of the modified activated carbon. Optimum modification process developed textural or surface functional groups properties of modified activated carbon that improved adsorption or binding capacity toward adsorbate or a particular species. In addition, the adsorption capacity of modified and corresponding activated carbon is compared.[3]

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Membrane localization of Rho GTPases is essential for their biological functions and is dictated in part by a series of posttranslational modifications at a carboxyl-terminal CaaX motif: prenylation at cysteine, proteolysis of the aaX tripeptide, and carboxymethylation. The fidelity and variability of these CaaX processing steps are uncertain. The brain-specific splice variant of Cdc42 (bCdc42) terminates in a CCIF sequence. Here we show that brain Cdc42 undergoes two different types of posttranslational modification: classical CaaX processing or novel tandem prenylation and palmitoylation at the CCaX cysteines. In the dual lipidation pathway, bCdc42 was prenylated, but it bypassed proteolysis and carboxymethylation to undergo modification with palmitate at the second cysteine. The alternative postprenylation processing fates were conserved in the GTPases RalA and RalB and the phosphatase PRL-3, proteins terminating in a CCaX motif. The differentially modified forms of bCdc42 displayed functional differences. Prenylated and palmitoylated brain Cdc42 did not interact with RhoGDIα and was enriched in the plasma membrane relative to the classically processed form. The alternative processing of prenylated CCaX motif proteins by palmitoylation or by endoproteolysis and methylation expands the diversity of signaling GTPases and enables another level of regulation through reversible modification with palmitate. [2]

C39H56N8O10S2

861.04

759.235632

1006592-64-8

172653583

Brown to orange solid powder

5

14

14

52

1580

0

CCN(CC)CC.C1=CC(=C(C=C1C(=O)NCCCCCCN=[N+]=[N-])C(=O)O)C2=C3C=CC(=[NH2+])C(=C3OC4=C2C=CC(=C4S(=O)(=O)[O-])N)S(=O)(=O)O

XOPKJMYTNXSBHS-UHFFFAOYSA-N

InChI=1S/C27H26N6O10S2.C6H15N/c28-19-9-7-16-21(17-8-10-20(29)25(45(40,41)42)23(17)43-22(16)24(19)44(37,38)39)15-6-5-14(13-18(15)27(35)36)26(34)31-11-3-1-2-4-12-32-33-30;1-4-7(5-2)6-3/h5-10,13,28H,1-4,11-12,29H2,(H,31,34)(H,35,36)(H,37,38,39)(H,40,41,42);4-6H2,1-3H3

3-amino-6-azaniumylidene-9-[4-(6-azidohexylcarbamoyl)-2-carboxyphenyl]-5-sulfoxanthene-4-sulfonate;N,N-diethylethanamine

Alexa fluor 488 azide ditriethylamine; 1006592-64-8

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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