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
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
- 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
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 (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 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
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 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
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.
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
- 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. Commercially available biopolymers can be supplied by customers or synthesized or ordered through Bio-Synthesis.
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.
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
- SPDP: Heterobifunctional cross linker. Commonly used for immunotoxins, antibody-enzyme
and enzyme-labeled DNA probes.
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
- Horseradish Peroxidase (HRP)
- Alkaline Phosphatase (AP)
- β-Galactorsidease Glucose Oxidase (GO)
- 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,
- 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
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
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’s needs, the more accurate your provided feedback will be. Providing us with your project’s details enables 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 (i.e. heterobifunctional or homobifunctional reagents), spacer is cleavable and if 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 containing 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 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, making the actual distance spanned by such linker arms less than expected. Instead, spacers containing 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 alkyl ether (PEO) chain. Bio-Synthesis offers several cross-linkers with PEO chains, such as thiol-binding homobifunctional reagents, heterobifunctional bases 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.