Lumiprobe Newsletter- Certificates of Analysis (CoA), Fluorescent Cyanine3 hydrazides, Cyanine3 amines , Cyanine5.5 hydrazides, Tech Questions and Citations
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Certificates of Analysis (CoA) on every webpage!
 
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Certificate of Analysis (CoA) is a document confirming structure and purity of each batch of material produced by Lumiprobe.

Lumiprobe proudly provides detailed certificates of analysis containing (depending on particular product) real NMR, UV and mass spectra, and HPLC chromatograms.  The data can be retrieved electronically from Lumiprobe.com.  The provided experimental data are useful in R&D work, and can be used to check the purity of the material.


Lumiprobe's new reagents  Cyanine3 and 5.5 hydrazides and amines
Cyanine3 hydrazide
http://www.lumiprobe.com/p/cy3-hydrazide
 
Cyanine3 hydrazide is a carbonyl-reactive dye, an analog of Cy3® hydrazide.

This reagent allows you to label various carbonyl-containing molecules such as antibodies and other glycoproteins after periodate oxidation, proteins which have undergone oxidative stress or deamination, or reducing saccarides. Cyanine3 is compatible with a number of fluorescent instruments.

Appearance:             red powder           
Molecular weight:     543.57
Molecular formula:   C30H40Cl2N4O
Solubility:     moderate solubility in water, good in polar organic solvents (DMF, DMSO, alcohols)

Quality control: NMR 1H and HPLC-MS (95%)%

Spectral properties

Excitation maximum, nm:     555
Extinction coefficient at excitation maximum, Lmol-1cm-1: 150000
Emission maximum, nm:     570
Fluorescence quantum yield:     0.31
                           
Cyanine3 amine
http://www.lumiprobe.com/p/cy3-amine

Cyanine3 amine is a functionalized cyanine dye containing a free amino group. Cyanine3 is an analog of Cy3®.

Amino group of this reagent can be conjugated with reactive groups such as NHS esters, carboxy groups (after carbodiimide activation), and epoxides.

The amino dye is supplied in salt form, and possesses some aqueous solubility.

Appearance:            red powder
Molecular  weight:   
627.73
Molecular formula:   C36H52Cl2N4O
Solubility:     moderate solubility in water, good in polar organic solvents (DMF, DMSO, alcohols)

Quality control:     NMR 1H, HPLC-MS (95%)

Spectral properties
Excitation maximum, nm:     555
Extinction coefficient at excitation maximum, Lmol-1cm-1:     150000
Emission maximum, nm:     570
Fluorescence quantum yield:     0.31

Cyanine5.5 hydrazide is a carbonyl-reactive near infrared dye. Hydrazides quickly react with carbonyl compounds, such as aldehydes and ketones, and form stable hydrazones. This reaction is useful for the preparation of labeled conjugates.

Carbonyl groups in biomolecules can form spontaneously after oxidative stress or deamination of proteins. Alternatively, aldehyde groups can be specifically generated by periodate oxidation of sugar residues of glycoproteins, for example antibodies. Antibodies can be oxidized by sodium periodate and labeled with Cyanine5.5 hydrazide to form Cyanine5.5 labeled antibodies. Since sugar residues in antibodies are located far from variable, epitope-binding site, this method is particularly good for the labeling of antibodies.

Cyanine5.5 is a near infrared fluorophore, an analog of Cy5.5®. It is particularly suitable for the applications which require low fluorescence background. The absorption and emission wavelength of the dye exhibit significant tissue penetration, so Cyanine5.5 is suitable for in vivo NIR imaging applications.


Appearance:            dark blue powder
Molecular                  669.73 weight:    
Molecular formula:   C40H46Cl2N4O
Solubility:         moderate solubility in water, good in polar organic solvents (DMF, DMSO, alcohols)

Spectral properties
Excitation maximum, nm:     673
Extinction coefficient at excitation maximum, Lmol-1cm-1:     209000
Emission maximum, nm:     707
Fluorescence quantum yield:     0.2

Cy® is a trademark of GE Healthcare..

Citations of interest !
Diabetes and insulin, improving solar energy,  drug delivery for cancer treatment & tumors,
microbiology In situ visualization of newly synthesized proteins

Would you like your paper featured on Lumiprobe citation webpage? Email: order@lumiprobe.com

In vivo imaging of GLP-1R with a targeted bimodal PET/fluorescence imaging agent

Brand, C.; Abdel-Atti, D.; Zhang, Y.; Carlin, S.D.; Clardy, S.M.; Keliher, E.J.; Weber, W.A.; Lewis, J.S.; Reiner, T.
 
Bioconjugate Chem. Just Accepted.
doi: 10.1021/bc500178d

Accurate visualization and quantification of β-cell mass is critical for the improved understanding, diagnosis, and treatment of both type 1 diabetes (T1D) and insulinoma. Here, we describe the synthesis of a bimodal imaging probe (PET/fluorescence) for imaging GLP-1R expression in the pancreas and in pancreatic islet cell tumors. The conjugation of a bimodal imaging tag containing a near-infrared fluorescent dye, and the copper chelator sarcophagine to the GLP-1R targeting peptide exendin-4 provided the basis for the bimodal imaging probe. Conjugation was performed via a novel sequential one-pot synthetic procedure including 64Cu radiolabeling and copper-catalyzed click-conjugation. The bimodal imaging agent 64Cu-E4-Fl was synthesized in good radiochemical yield and specific activity (RCY = 36%, specific activity: 141 μCi/μg, >98% radiochemical purity). The agent showed good performance in vivo and ex vivo, visualizing small xenografts (<2 mm) with PET and pancreatic β-cell mass by phosphor autoradiography. Using the fluorescent properties of the probe, we were able to detect individual pancreatic islets, confirming specific binding to GLP-1R and surpassing the sensitivity of the radioactive label. The use of bimodal PET/fluorescent imaging probes is promising for preoperative imaging and fluorescence-assisted analysis of patient tissues. We believe that our procedure could become relevant as a protocol for the development of bimodal imaging agents.

Photoresponsive protein–graphene–protein hybrid capsules with dual targeted heat-triggered drug delivery approach for enhanced tumor therapy

Hu, S.H.; Fang,R.H.; Chen,Y.W.; Liao, B.J.; Chen, I.W.; Chen, S.Y.
Advanced Functional Materials, in press.
DOI
: 10.1002/adfm.201400080


A novel photo-responsive protein–graphene–protein (PGP) capsule that doubles as a photothermal agent with core/shell structure is constructed by anchoring reduced graphene oxide nanosheets on one-component protein (lactoferrin) shell through a double emulsion method. PGP capsules can transport fully concealed hydrophilic anticancer cargo, doxorubicin (Dox), with a large payload (9.43 μmol g-1) to be later unloaded in a burst-like manner by photo-actuation triggered by near-infrared irradiation. Being biocompatible yet with a high cancer cell targeting efficiency, PGP capsules have successfully eradicated subcutaneous tumors in 10 d following a single 5 min NIR irradiation without distal damage. Besides, the photochemothermal therapy of PGP capsules eradicates tumor cells not only in the light-treating area but also widely light-omitted tumor cells, overcoming the tumor recurrence due to efficient cell killing efficacy. These results demonstrate that the PGP capsule is a potential new drug delivery platform for local-targeting, on-demand, photoresponsive, combined chemotherapy/hyperthermia for tumor treatment and other biomedical applications.


Near-infrared harvesting transparent luminescent solar concentrators

Zhao,Y.; Meek, G.A.; Levine, B.G.; Lunt, R.R.
Advanced Optical Materials
doi: 10.1002/adom.201400103

Near-infrared (NIR) harvesting transparent luminescent solar concentrators (TLSC) with non-tinted transparency are demonstrated by exploiting the excitonic nature of organic luminescent salts that provide perfectly tuned NIR-selective absorption and even deeper NIR emission. NIR TLSCs provide an entirely new route to more aesthetically pleasing light harvesting systems that can be widely deployed in energy scavenging windows and displays.


In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry.

Hatzenpichler, R.; Scheller, S.; Tavormina, P.L.; Babin, B.M.; Tirrell, D.A.; Orphan, V.J.
Environmental Microbiology, in press.
doi: 10.1111/1462-2920.12436

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level.


Tech Support: Lumiprobe offers FREE tech support before or after you order.
A few questions:

Can Cyanine5 hydrazide  label free carbonyl or free carboxyl group?
If the Cy5 hydrazide can label free carbonyl or free carboxyl group, would you please provide protocol?

Cy5 hydrazide can only label free carbonyl groups, not carboxyl groups.

Here is a protocol:

1. Dissolve the carbonyl compound (such as protein bearing carbonyl group) in aqueous buffer (e.g acetate) with pH 5.5. Protein concentration of around 10 mg/mL is recommended for optimal labeling.
2. Dissolve 1 mg of Cy5 hydrazide in 50 uL of DMSO.
3. Add 5 eq. of hydrazide solution to a solution of carbonyl compound so that final concentration of DMSO is around 10%.
4. Vortex and keep for 1 h at room temperature.
5. Purify the conjugate by gel filtration, electrophoresis, or HPLC.

I am writing to look for your help with labelling Eudragit® RS polymer. I plan to administer the labelled polymer to mice and then do some animal in vivo imaging experiment using optical microscope. Actually I have not got any idea on labelling this polymer as it only contains quaternary amino group and ester group. Your kind help would be much appreciated and I am looking forward to your reply.”
    
Hydrazides and amines have reactivity towards esters. Reactivity of the former is higher. We can recommend trying Cyanine5 hydrazide to label this polymer. The reagent should be mixed with the polymer in appropriate solvent (most probably, mixture of water with DMSO or DMF) in the presence of buffer (pH 8-9), and hydrazide solution in DMSO/DMF should be added. Some extended time is expected to complete the reaction. Purification can be based on gel filtration or other method appropriate for the polymer.  Believe it should work. Hope this can be helpful!

I have a couple questions regarding your Cy5 amine (cat. #130C0) – what is the solubility limit of this reagent in aqueous solutions? Also, once reconstituted in solution, is the reagent stable (in terms of fluorescence, aggregation, etc.)?  Is there a specific buffer/pH you could recommend for reconstituting this reagent?     Also, once reconstituted, would you recommend storing it frozen in aliquots, or just storing it at 4deg?    Thanks for your help!

This reagent has moderate and pH dependent solubility in aqueous solutions. I guess it is around tens of mM in water, but we have not determined it. We can measure it if it is an important parameter for you.   
   
Stability depends on pH. In neutral and slightly acidic solutions, it is pretty stable. It is not stable in alkaline solutions, because nucleophiles react with Cy5. However, it is stable enough for the conjugation of amino group.   
   
You could dissolve it in a buffer with pH 6 or below - for example, citrate or acetate buffer should work well. Solutions will be most stable if frozen. Most stable form is solid dye, but guess that frozen solution in acidic buffer can store for many months.   


I am looking for Cyanine5.5 for animal imaging purpose.  However you have Cyanine5.5 alkyne, Cyanine5.5 NHS ester, Cyanine5.5 azide, Cyanine5.5 carboxylic acid, and Cyanine5.5 maleimide. I don’t know which Cyanine5.5 is good for my purpose.  Could you please give me an advice?

Thank you for contacting Lumiprobe!

All these reagents contain same fluorophore, Cy5.5, which can be used for the imaging. What kind of imaging would you like to do? Generally you will need a labeled molecule of interest (protein, peptide, etc) containing Cy5.5 label. All these reagents are intended for different labeling chemistries.

NHS ester and maleimide are for the labeling of peptide NH2 groups and thiol groups correspondingly - most probably, one of them is good for you.


On the Lumiprobe.com website there is much information on each reagent, including info on spectral properties, often a graph of absorption and emmission spectra, a link to download the MSDS and other information.

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