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Enzyme Labeling Bioconjugation
Enzyme Labeling Bioconjugation

Custom Enzyme Conjugation and Modification

Bio-Synthesis offers custom enzyme conjugation and modification services to be used as signal generating molecules. Enzymes such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (ALP), can be attached to antibodies, oligonucleotides, peptides or proteins to act as signal molecules in various applications.

Our unique and unrivalled expertise in bioconjugation chemistries offers you the best quality enzyme biopolymer conjugates in the industry. Through the use of different coupling chemistries, Bio-Synthesis can maintain the activity of the antibody by selectively conjugate enzyme to antibody without interfere with the antigen binding site. In addition, non-enzymatic fluorescent labels and biotin can be coupled to specific antibodies, proteins, and peptides as a means of signal generation.

Enzymes we use

  • Horseradish Peroxidase (HRP)
  • Alkaline Phosphatase (ALP)
  • Alkaline Phosphatase Amplification (AMP)
  • Penicillinase (PNC)
  • Biotin-Avidin System
  • alpha-D-Galactorsidse
  • Glucose-t-Phosphate Dehydrogenase
  • Other Enzyme Innunoconjugates
  • Nonenzyme protein immunoconjugates

Our custom enzyme bioconjugation services include experimental design, development of protocols and procedures for the preparation of conjugates to be used in various detection and assay systems. Contact our Technical Service Center at 800.220.0627 or contact us online with your detail project specifications, a project manager will be assigned to help you with design and develop an appropriate synthetic method for your specific needs.

Enzyme Immunoconjugates

Reporter enzyme conjugated probes are widely used in many different experimental applications because of their ability to be conjugated to many different kinds of macromolecules and the availability and variability of substrates. Enzymes are most commonly used to detect protein via direct and indirect antibody detection strategies or as a nucleic acid hybridization probes needed in sensitive assay, and the detection methods. These reporter enzymes are used extensively in molecular biology because they have particular characteristics that allow visual and spectrophotometric detection of these immune complexes. In spite of significant advances in the detection of fluorescent and luminescent labels, enzymes continue to be the most sensitive reporter groups. For most applications, direct enzyme labels offer the best overall performance, with highest sensitivity, least background, and rapid detection.

The benefits of using enzyme reporter probes

  • High sensitivity – The signal output can be easily detected, and therefore low concentrations of target proteins can be identified. Additionally, methods of signal amplification are available that significantly increase the number of enzyme molecules to the site of the target protein. Finally, enzyme reporters exhibit rapid turnover, which increases the amount of substrate that a single enzyme converts during a given unit of time.
  • Long shelf life – The enzymes are quite stable when stored properly, and while the enzyme substrate is light-sensitive, the enzyme itself is not sensitive to degradation by ambient light.
  • Output versatility – Substrates that yield either chromogenic, chemiluminescent or fluorescent output are available for the most common enzyme probes.
  • Although these benefits demonstrate the versatility and convenience of enzyme probes, there are limitations to their use that should be considered when choosing the appropriate type of detection probe:

  • Large size – Enzyme reporters are considerably larger than organic fluorescent compounds (e.g., FITC, TRITC, AMCA) and therefore may interfere with the biological function of proteins to which they are conjugated.
  • Substrate requirement – Enzyme probes require the addition of a substrate for protein detection, and depending on the substrate used, this reaction can be sensitive to environmental conditions (e.g., light, temperature) and ambient light.
  • Endogenous interference – The enzymes used to detect target proteins in a sample are often expressed in the experimental system used (e.g., tissues, cells), which will also process the substrate and yield nonspecific background signal unless inhibited.

Horseradish peroxidase

Horseradish peroxidase (HRP) catalyzes the transfer of two electrons in a substrate hydrogen peroxide to produce an oxidized substrate and water.  For detection of protein, HRP substrates (listed in the table below) are designed to generate a signal chromogenic, chemiluminescent or fluorescent oxidation.  HRP has a molecular weight of 40,000, which is relatively low compared with other enzyme conjugates.  This small size allows greater penetration in tissues and cells of the sample and reduces the likelihood of interfering with the function of the protein conjugate.  HRP also has four lysines available to bowing, which improves the efficiency crosslinking to a protein of interest.

HRP has a high turnover and produces abundant reaction products in a short time at physiological pH (7.6).  IgG conjugated with HRP was higher than alkaline phosphatase and-galactosidase conjugates due to their higher specific enzyme activity (more than the HRP / mole of antibody) and immune reactivity (lower steric hindrance due to the size of HRP).

A major problem associated with the use of HRP is nonspecific staining that result from the endogenous peroxidase activity in some tissues. Cryostat sections often contain a significant amount of endogenous peroxidase activity, although commercial peroxidase inhibitors are available to reduce or eliminate endogenous peroxidase activity.  Enzyme HRP is the label of choice for staining of paraffin sections of the structure, a process that inhibits the endogenous activity.

A second drawback to HRP is its susceptibility to degradation by micro-organisms and antibiotics used to combat them.  Sodium azide is a potent inhibitor of HRP, but the enzyme can be stored in 0.01% thimerosal.  HRP is also inhibited by cyanides, sulfides and azides.  A disadvantage of HRP is mutagenic or carcinogenic products of the reaction of some substrates of horseradish peroxidase.  If none of these problems is a big concern, other enzymatic markers may be preferred.

Alkaline phosphatase

Alkaline phosphatase (AP) is a widespread family of enzymes that hydrolyze nucleotide phosphates and proteins.  Optimal enzyme activity occurs at a pH of 9.0 to 9.6, these enzymes are activated by divalent cations and inhibited by cysteine, cyanide, arsenate, inorganic phosphate and divalent cation chelators such as EDTA.

There are two forms of alkaline phosphatase in mammals, one of the forms distributed in many tissues and the other is found in the gut.  Both forms are affected differently by inhibitors of heat and inactivators.  The use of 1 mM levamisole in the substrate buffer to inhibit endogenous phosphatase of tissues.  activity of intestinal alkaline phosphatase can be inhibited by treatment of sections before staining with 20% acetic acid at 4 ° C for 15 seconds (or 2.3% periodic acid for 5 minutes), followed by 0.02% potassium borohydride for 2 minutes.

Calf intestinal alkaline phosphatase is perfect for applications where high levels of endogenous peroxidase-cons use of conjugates with HRP, the cryostat, where the peroxidase inhibitors are ineffective.

When used as a label, calf intestinal alkaline phosphatase (molecular weight 140 000) offers several advantages over other enzymes.  Because the reaction rate is linear, the sensitivity can be improved by the reaction to continue for a long time.  The activity of intestinal alkaline phosphatase calf is not affected by exposure to antibacterial agents such as sodium azide or thimerosal, so it can be stored for a long time in sterile environments.  Because the endogenous activity of intestinal alkaline phosphatase can be inhibited by mM levamisole, enzyme-labeled antibodies can be used as markers for many different tissues.

Glucose oxidase

Glucose oxidase is an enzyme isolated from Aspergillus Niger catalyzing the oxidation of beta-D-glucose to produce hydrogen peroxide and gluconic acid.  The glucose oxidase is a glycoprotein dimer with a molecular weight of 160,000.  Inhibitors of glucose oxidase include Ag+, Hg2+ , Cu2+ , p-chloromercuribenzoate and phenylmercuric acetate.

The glucose oxidase is often the label of choice for samples with high endogenous peroxidase or alkaline phosphatase, as no activity of glucose oxidase in endogenous mammalian tissues.  It is important to choose a glucose oxidase with low catalase activity, but because catalase destroys hydrogen peroxide produced in the reaction.

β-galactosidase

β-galactosidase is an enzyme isolated from E. coli that is capable of hydrolyzing a variety of galactopyranoside derivatives, which produce both water-soluble and water-insoluble products. NaCl is an activator and Mg2+ is a cofactor of this enzyme, and the optimum pH for β-galactosidase is 7.0-7.5. For immunohistochemical staining, potential interference from endogenous enzyme can be overcome by embedding the sample with paraffin.

β-Galactosidase is sensitive and demonstrates no endogenous activity in mammalian cells, and therefore it is useful in applications where endogenous enzyme activity is a persistent problem. β-Galactosidase has been successfully coupled by a variety of crosslinkers to IgG fragments, whole immunoglobulins and insulin. One disadvantage of β-galactosidase is a lack of substrate variety.

Biotin

The attachment of biotin to biomolecules is an important laboratory technique. Biotin binds to the tetrameric avidin proteins, including streptavidin and neutravidin, with exceptionally high affinity, and this interaction is exploited in various applications such as western blotting, immunohisthochemistry and ELISA. Supplied antibody will be labeled with a long spacer arm biotin derivative follow by appropriate purification method. The final average ratio of antibody to biotin will be determined by performing HABA biotin quantification assay.

Custom Enzyme Biopolymer Conjugation Services

Our enzyme bioconjugation services use various types of crosslinking chemistries. If you can’t find what you need, please contact us online with your detail project descriptions.

Service Portfolios

  • Horseradish Peroxidase (HRP)-Conjugated Antibodies
  • Fab'-HRP Conjugates
  • Fab-HRP Conjugates
  • Half Antibody - HRP Conjugate
  • Alkaline Phosphatase (ALP)-Antibodies Conjugates
  • Alkaline Phosphatase (ALP)-Oligo Conjugates
  • Protein-Horseradish Peroxidase (HRP) Conjugates
  • Protein-Alkaline Phosphatase Conjugate (ALP)
  • Horseradish Peroxidase (HRP)-Peptide Conjugates
  • Alkaline Phosphatase (ALP)-Peptide Conjugates
  • Enzyme-Biotin Labeling
  • Enzyme-Nanoparticle Bioconjugates

Sample Submission Requirement

Biomolecule supplied by customers should be sufficiently pure. Please provide 5 mgs of starting material with the necessary data for purity assessment. Commercial available biopolymers can be supplied by customers or synthesize or 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.

Chemistry

Enzyme can be modified to contain reactive groups to react with other preactivated small molecule and biomolecule with chemical reactive groups such as amine, thiol, carboxylate, hydroxyl, aldehyde and ketone, etc.

Typical preparation of enzyme conjugates are:

  • Glutaraldehyde: Homobifunctional crosslinker containing an aldehyde functional group at both ends of a 5-carbon chain will react with amines to form secondary amine linkage.
  • Periodate Oxidation: Enzyme such as GO and HRP can be oxidize with periodate to create reactive aldehyde residues for conjugation.
  • SMCC: Herobifunctional corsslinker or water-soluble analog sulfo-SMCC can be used to activate enzymes through their amines to form amide bonds.
  • Hydrazide: Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages.
  • SPDP: Heterobifunctional cross linker. Commonly used for immunotoxins, antibody-enzyme and enzyme-labeled DNA probes.

Service Specification

Our enzyme conjugates are prepared by techniques that yield an approximate 1:1 ratio of enzyme to protein of interest, unless otherwise specified by the customer. After standard desalting, or purification, a small percent of heterogeneous products containing single or multi-site conjugate per molecule may exist.

Material :

  • Enzyme
    • Horseradish Peroxidase (HRP)
    • Alkaline Phosphatase (AP)
    • β-Galactorsidease Glucose Oxidase (GO)
  • Biopolymers
    • 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 of enzyme with the crosslinking reagent, 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. Additional purification technique such as stirred cell filtration, tangential flow filtration (TFF), gel filtration chromatography may also be used to either remove excess reagent or isolate and characterized the cross-linked product. 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.

Deliverable:

2-3 weeks

Ordering and Submitting Requests 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.  Provide us with your project details will enable to us to recommend the best reagents to use for your 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 aremore 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 and length of the spacer arm 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.

Within 3-5 days upon receiving your project scope, we will provide you an appropriate quotation. An order can be placed with PO (Purchase Order) or major credit cards ( ). Your credit card will be billed under Bio-Synthesis, Inc.