Inside this newsletter
- Useful for Manufacturing Oligonucleotide Therapeutics
- Citations of Interest
- Scientist's Questions - Increase your knowledge and ideas!
- Lumiprobe New Years greeting!
Lumiprobe reagents reduce cost, increase productivity and improve quality of research and production. Thanks to all ! Save 5% use discount code: biocrowd
Lumiprobe offers over 80 reagents for peptide, oligonucleotide, amine, alkyne binding - useful for Manufacturing Oligonucleotide Therapeutics!
In 2014, whether researching, creating, or manufacturing new drugs and treatment, Lumiprobe reagents were giving results in drug development research. Click chemistry was used with peptide-based nanoparticles in vivo, comparing the advantages between linear and cyclic peptides for intracellular delivery, process improvements for the economicÂ production,Â peptide characterization , detecting and controlling oligonucleotide impurities, and exploring the development of peptides for diagnostic applications.Â Novel oligonucleotide and peptide therapies were also enhanced when Lumiprobe's reagents were included.
Dye NHS esters - amine-reactive cyanine activated esters for the labeling of proteins, peptides, and other molecules
Sulfo NHS esters - water soluble sulfo-Cyanine3 SE activated ester for amino-biomolecule labeling.
Azides - fluorescent biomolecule labeling through Click Chemistry
Alkynes - alkyne dye,Â Useful for both copper-catalyzed, and copper-free Click Chemistry.
Maleimides - bright and photostable thiol-reactive dye for protein labeling
Amines - fluorophores with free amino group (amino-dye). It can be conjugated with NHS esters, alkynes, carboxy groups (after activation), and epoxides.
Hydrazides - dyes with a reactivity for carbonyl groups (aldehydes and ketones) activated by acid.Â It can be used for the labeling of glycoproteins (i.e. antibodies) after periodate oxidation of sugar moieties.
Citations of Interest !
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Over 100 papers sorted by date or reagent are available for your review.
In Site-Selective Protein Immobilization by Covalent Modification of GST Fusion Proteins.
Zhou, Y.; Guo, T.; Tang, G.; Wu, H.; Wong, N.-K.; Pan, Z.
Bioconjugate Chemistry, 2014 doi: 10.1021/ bc50 0347b
EGF receptor-targeting peptide conjugate incorporating a near-IR fluorescent dye and a novel 1,4,7-triazacyclononane-based 64Cu(II) chelator assembled via click chemistry.
The immobilization of functional proteins onto solid supports using affinity tags is an attractive approach in recent development of protein microarray technologies. Among the commonly used fusion protein tags, glutathione S-transferase (GST) proteins have been indispensable tools for protein–protein interaction studies and have extensive applications in recombinant protein purification and reversible protein immobilization. Here, by utilizing pyrimidine-based small-molecule probes with a sulfonyl fluoride reactive group, we report a novel and general approach for site-selective immobilization of Schistosoma japonicum GST (sjGST) fusion proteins through irreversible and specific covalent modification of the tyrosine-111 residue of the sjGST tag. As demonstrated by sjGST-tagged eGFP and sjGST-tagged kinase activity assays, this immobilization approach offers the advantages of high immobilization efficiency and excellent retention of protein structure and activity.
Viehweger, K.; Barbaro, L.; Garcia, KP; Joshi, T.; Geipel, G.; Steinbach, J.; Stephan, H.; Spiccia, L.; Graham, B. Bioconjugate Chem., 2014, 25(5), 1011–1022. doi: 10.1021/ bc50 01388
A new Boc-protected 1,4,7-triazacyclononane (TACN)-based pro-chelator compound featuring a “clickable” azidomethylpyridine pendant has been developed as a building block for the construction of multimodal imaging agents. Conjugation to a model alkyne (propargyl alcohol), followed by deprotection, generates a pentadentate ligand, as confirmed by X-ray crystallographic analysis of the corresponding distorted square-pyramidal Cu(II) complex. The ligand exhibits rapid 64Cu(II)-binding kinetics (>95% radiochemical yield in <5 min) and a high resistance to demetalation. It may thus prove suitable for use in 64Cu(II)-based in vivo positron emission tomography (PET). The new chelating building block has been applied to the construction of a bimodal (PET/fluorescence) peptide-based imaging probe targeting the epidermal growth factor (EGF) receptor, which is highly overexpressed on the surface of several types of cancer cells. The probe consists of a hexapeptide sequence, Leu-Ala-Arg-Leu-Leu-Thr (designated “D4”), followed by a Cys-β-Ala-β-Ala spacer, then a β-homopropargylglycine residue with the TACN-based chelator “clicked” to its side chain. A sulfonated near-infrared (NIR) fluorescent cyanine dye (sulfo-Cy5) was introduced at the N-terminus to study the EGF receptor-binding ability of the probe by laser-fluorescence spectroscopy. Binding was also confirmed by coimmunoprecipitation methods, and an apparent dissociation constant (Kd) of ca. 10 nM was determined from radioactivity-based measurements of probe binding to two EGF receptor-expressing cell lines (FaDu and A431). The probe is shown to be a biased or partial allosteric agonist of the EGF receptor, inducing phosphorylation of Thr669 and Tyr992, but not the Tyr845, Tyr998, Tyr1045, Tyr1068, or Tyr1148 residues of the receptor, in the absence of the orthosteric EGF ligand. Additionally, the probe was found to suppress the EGF-stimulated autophosphorylation of these latter residues, indicating that it is also a noncompetitive antagonist.
Size- matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.
Burnham- Marusich, A.R.; Plechaty, A.M.; Berninsone, P.M.
ELECTROPHORESIS, 2014, 35, 2621–2625. doi: 10.1002/elps.201400241
Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In “Click-DIGE”, posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs.
Tech Support: Lumiprobe offers FREE tech support before or after you order.
Questions - Increase your knowledge and ideas!
I have few questions about the click-chemistry labeling and need suggestions. In the protocol for Click-Chemistry Labeling of Oligonucleotides and DNA,
Do you provide the service which conjugate peptide to cy5 NHS ester?
1.Â Triethylammonium acetate buffer (final concentration 0.2 M, pH 7.0) was used. Does it matter that types of salts and pH of buffer effect the labeling efficiency? Can PB buffer be used?
2.Â Azide was dissolved in DMSO and 1.5 * (DNA concentration), is it OK that azide was dissolved in water? In my experiment, azide was connected to peptide and had a good water solubility. Is it 50% DMSO necessary for the performance of click-chemistry labeling?
3.Â Degass steps were performed during the procedure. Does it sensitive to oxygen?
4.Â Precipitating the conjugate with acetone or ethanol was preformed to remove excess small molecules. Can Amicon ultra 3K device (Millipore) for buffer changing get the same result?
5.Â Can reaction yield get >90%? Will +4 Centigrade lower the yield?
Thank you for your email!
1. In practice, Click Chemisty displays little dependence on the composition of buffer. It should not contain free thiols (i.e. no DTT or mercaptoethanol). The reaction can be carried out at pH of at least 4 to 10. I believe phosphate will work fine.
2. You can dissolve your azide in water without any issues. The protocol uses DMSO because it is optimized for non water-soluble azides.
3. Yes, the reaction is sensitive to oxygen. Alkyne dimers can form upon oxidation. Moreover, hydroxyl radicals are formed upon oxidation of Cu(I) catalyst, and they can damage biomolecules, especially DNA/RNA. We recommend to degass the solution.
4. Any purification will work, precipitation is the easiest for oligo labeling. You can desalt the reaction.
5. Yes, yields may exceed 90%. Lowering temperature to +4 Centigrade will not harm, but probably will extend the required conjugation time.
Please do not hesitate to contact Lumiprobe if you have more questions.
Actually, I do have one more question about NHS-ester labeling. The optimal pH value for modification is 8.3-8.5. In most cases I saw, the pH value suggested is 7.4. For example, SDA (pierce, product 26167) I used for proteins labeling is used in PBS. Is there any difference between DNA labeling and protein labeling? The pKa value of primary amines is the key factor?
Optimal pH for NHS ester labeling is indeed 8.3-8.5. pH value of 7.4 can be used if you need to label preferably N-terminus of a protein rather than side chains of lysine. The terminal amino-group is less basic than lysine amine, and it is less protonated at this pH than that of lysine. Therefore, it reacts preferably with the NHS ester. At higher pH ranges, both amino groups are non-protonated, and since lysine amines are more reactive, they react first.
If you need to label protein lysines or aminolink-DNA, use pH 8.3-8.5.
Unfortunately, Lumiprobe does not make labeled peptides and does not perform conjugation. However, Lumiprobe can supply you or service company of your choice with our fluorescent reagents. The labeling protocol is simple enough to be performed by you. If you have peptide purification facilities such as HPLC -Â if you work with peptides, you likely have it). If you prefer labeling as a service, there are local companies who do it - Lumiprobe can supply our reagent to this company. This will help save money on reagents.
Why use Lumiprobe's fluorescent dyes such as Cyanine5 NHS ester?
Cyanine5 fluorophore emission has maximum in red region, where many CCD detectors have maximum sensitivity, and biological objects have low background. Dye color is very intense, therefore quantity as small as 1 nanomol can be detected in gel electrophoresis by naked eye.
* high quality,Â ideal for very cost-efficient labeling
* works well in organic solvents for small molecule labeling of soluble proteins,
peptides and oligonucleotides
* Water-soluble sulfo-Cyanine 5 NHS ester for sophisticated cellular & protein targets
* Compatible with various instrumentation including fluorescent microscopes,
imagers, scanners, and fluorescence readers.
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Lumiprobe New Year's greeting!
Thank you for integrating Lumiprobe fluorescent probes into your work in 2014!
Restock your lab in January and save 5%.
Use discount code: biocrowd
Restock your lab or begin a new project - Lumiprobe offers you a new year's email discount of 5%. Use discount code: biocrowd
If you need something not found in Lumiprobe's catalog - ask!
Lumiprobe offers to help with your research, and create a custom probe for you at no extra charge! Reagents can often be customized for your needs at price schedule similar to Lumiprobe's other reagents. Ask Lumiprobe's tech support - you'll receive attention to your research, and ideas on what or how to do the click chemistry reaction. Lumiprobe's website offers instantly downloadable Certificate of Analysis (CoA) detailing meticulous quality control, real NMR, UV and mass spectra, and HPLC chromatograms.