Fluorescent Dye Labeling

Custom Biopolymer Labeling

There are many ways to label biopolymers. The choice is determined by the technique you are going to use in your experiment. Detection of probes or labels usually takes one of three general forms: spectrophotometric, radiosensitive detectors, or indirectly through another labeled substance. Spectral probes can be of two types, chromogenic or fluorescent. Chromogenic label typically are reserved for non-covalent staining of gross structural features within cells and tissues, as these are present at relatively high concentration. The sensitivity of visible wavelength dye often is not good enough to provide detectability for low-concentration antigens or low-copy proteins. Even if a protein is covalently modified with a chromogen, the number of associated dye molecule needed to detect it just through its absorbance properties could be prohibitively large to make it visible

Fluorescent molecules, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Protein, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissue s for the presence of particular antigens, and detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more than one labeled molecule- each tagged with a different fluorophore can be used at the same time to detect two or more target molecules.

Fluorescent labeling is the process of covalently attaching a fluorophore to another molecule, such as a protein or nucleic acid. This is generally accomplished using a reactive derivative of the fluorophore that selectively binds to a functional group present in the target molecule. The most commonly labeled molecules are antibodies, which are then used as specific probes for the detection of a particular target. Fluorescent labeling can be applied to a wide variety of detection systems and allows sensitive and quantitative measurement.

Whether you are labeling proteins and antibodies for immunofluorescence, nucleic acids for in situ hybridization, or lipids for membrane studies, Bio-Synthesis will help you find the right conjugation product.

Custom Biomolecule Labeling Services

Service Portfolios

• Oligonucleotide Labeling
• Peptide Labeling
• Antibody Enzyme Labeling
• Antibody Fluorescent Dye Labeling
• Half antibody Labeling through Thiol
• Protein Labeling
• Enzyme l\Labeling
• Oligo redox Labeling
• Organometalic Compounds
• Antibody Gold Labeling
• Oligo qDot Labeling

Sample Submission Requirement:

Biomolecule supplied by customers should be sufficiently pure. Please provide 1-3 mgs of starting material with the necessary data for purity assessment. Commercially available biopolymers can be supplied by customers or synthesized and ordered through Bio-Synthesis.

Price

Price varies based on the project specifications. Our service includes materials and labor for conjugation only! Price does not include the cost of biopolymer synthesis or order through Bio-Synthesis from a commercial vendors and, if deemed necessary, biopolymer modification to introduce additional functional groups, extra linkers, spacers. Please contact us for a quote.

Discount: 15 % discount pricing applies to additional conjugates ordered at the same time.

Chemistry

Coupling of preactivated label and biomolecule with chemical reactive groups such as amine, acid, aldehyde, hydrazine, hydroxyamine, maleimide/alkyl halide, and sulfhydryl functional group.

Service Specification: Our reporter labeling services are prepared by techniques that can allow site specific conjugation. Labeling ratio can be optimized at client's request. After standard desalting, or purification, a small percent of heterogeneous products containing single or multi-site conjugate per molecule may exist.

Material:

Biomolecule we use to label with various dyes, tags or probes are:

• Protein: Enzyme, antibodies, antigens, cell adhesion molecules
• Peptides: Synthetic polypeptides
• Saccharides: Sugars, oligosaccharides and polysaccharides
• Lipids: Fatty acids, phospholipids, glycolipids and any fat-like substances.
• Ligands: Hormone receptors, cell surface receptors, avidin and biotin, small molecules
• Labels: Fluorescent dyes, infrared-absorbing and UV-Vis absorption chromophores, nonradioactive labels
• Nucleic acids and nucleotides: DNA, RNA, PNA, nucleic acid analogs and genomic DNA
• Synthetic polymers: PEG, Nanoparticles, gold particles, dendrimers, dendron, PAMAM
• Others: Conjugated or mixtures of any the above
• Solid Supports: agarose, glass plates, membrane, beads

Procedure: After labeling, final conjugates must first be isolated from excess or unreacted reagent by gel filtration or dialysis. In many cases, simple dialysis may suffice to remove unreacted reagent from the reaction solution. Final concentration and labeling ratio determination (if applicable).

Optional services such as gel filtration chromatography may also be used to either remove excess reagent or isolate and characterized the cross-linked product. With exception of dialysis, if the protein/antibody is significantly larger (>3-fold) than the modifying or coupling reagent. For reagents (mostly protein and other biological molecules) that are similar in size or larger than the antibody, one must resort to other purification techniques such as affinity chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography.

Cross-linked target molecule may then be further characterized by biochemical or biophysical techniques. Once the product has been purified, it may be subject to many different types of studies including spectroscopic (MALDI-TOF, ESI, LC-MS Fluorescence), electrophoresis, immunochemical biochemical, enzymatical analysis. QC (quality control) and QA (quality assurance) procedures are also followed independently to offer you double guarantee for the highest quality possible of every delivered conjugates. Moreover, our dedicated technical account managers will guide your project through every step of the process and constantly keep you informed of the latest project progress.

Links and Resources

Reference/Citing:

1. Upadhyay, A. et al.: Immunological Response to Peptide Nucleic Acid and its Peptide Conjugate Targeted to Transactivation Response (TAR) Region of HIV-1 RNA Genome; Oligonucleotides. 18(4) 2008.
2. Olejnik, J. et al.: Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS; Nucleic Acid Researech. 27 (23) 1999.
3. Pai, S. et al.: Proximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores; Nucleic Acid Research. 33(18) 2005.

Visit our literature vaults for more references and citings.

Ordering and submitting a request for Bioconjugation Services



For us to better understand your customized project, please complete our Bioconjugation Service Questionnaire. The more our chemists understand your project needs, the more accurate feedback we will be able to provide you.  Providing us with your project details will enable us to recommend the best reagents to use for your own project.  The most useful and readily available tools for bioconjugation projects are cross-linking reagents. A large number of cross-linkers, also known as bifunctional reagents, have been developed.  There are several ways to classify the cross-linkers, such as the type of reactive group, hydrophobicity or hydrophilicity, and the length of the spacer between reactive groups.  Other factors to consider are whether the two reactive groups are the same or different (for example, heterobifunctional or homobifunctional reagents), whether the spacer is cleavable, and whether the reagents are membrane permeable or impermeable.  The most accessible and abundant reactive groups in proteins are the ϵ-amino groups of lysine.  Therefore, a large number of the most common cross-linkers are amino selective reagents, such as imidoesters, sulfo-N-hydroxysuccinimide esters, and N-hydroxysuccinimide esters.  Due to the high reactivity of the thiol group with N-ethylmaleimide, iodoacetate and a-halocarbonyl compounds, new cross-linkers have been developed that contain maleimide and a-carbonyl moieties.  Usually, N-alkylmaleimides are more stable than their N-aryl counterparts.

 

In addition to the reactive groups on the cross-linkers, a wide variety of connectors and spacer arms have also been developed.  The nature of the spacer arm, and also the length, play an important role in the functionality.  Longer spacer arms are generally more effective when coupling large proteins or those with sterically protected reactive side-chains.  Other important considerations are the hydrophobicity, hydrophilicity, and the conformational flexibility.  Long aliphatic chains generally fold on themselves when in an aqueous environment, which makes the actual distance spanned by such linker arms less than expected.  Instead, spacers that contain more rigid structures (for example, aromatic groups or cycloalkanes) should be used.  These structures, however, tend to be very hydrophobic which could significantly decrease the solubility of the modified molecules or even modify some of their properties.  In such cases, it is recommended to choose a spacer that contains an alkylether (PEO) chain.  Bio-Synthesis offers several cross-linkers with PEO chains, such as thiol-binding homobifunctional reagents, heterobifunctional based, and their derivatives.

Once the project scope has collected, we will provide an appropriate quotation within 3-5 days. Orders can be placed with either a PO (Purchase Order) or credit card. We accept POs and major credit cards ( ). Your credit card will be billed under 'Bio-Synthesis, Inc.' Click here to download our credit reference form. For international orders, we must apply the full charge at the time of the order is placed. In the unlikely event that any given order cannot be filled, our guarantee will take the form of a full refund.