AAT Bioquest's substrates are a component of many of our kits, and are also available as standalone items. Spanning a wide spectral range, from the bright Amplite®
Blue to the long-wavelength Amplite®
IR, these substrates fluoresce in the presence of the peroxidase enzyme
and H2O2. This series has a high signal-to-noise ratio and is very stable. Each substrate demonstrates useful features; Amplite®
Blue does not photobleach, for example, and Amplite®
IR has both excellent water solubility and is pH-independent from pH 4-10.
Table 1. Amplite® Substrates For Fluorogenic Substrates
Enzymes act as a regulatory catalyst for the majority of biochemical reactions that facilitate cell function, including all aspects of cellular metabolism. They do so by lowering the energy barrier necessary for a chemical reaction to occur. Enzymes are present throughout the cell, some sequestered within organelles such as the mitochondria, or free in the cytoplasm. Upon reaction with a suitable substrate, enzymes change shape to form a new complex. This change, when used during experiments, can provide a signal to researchers that a reaction has occurred, as well as potentially improving the specificity of the reaction itself. Amplite®
enzyme-based assays provide a sensitive and simple method for the quantitation of a diverse array of analytes, including dehydrogenases
, and other enzyme groups. Learn More
Enzyme activity assays are the primary tool for studying enzyme kinetics and inhibition. AAT Bioquest offers a full line of enzymatic activity kits for targets including hydrolytic enzymes, proteases, and other enzyme families.
Click the button on the right to view or download our PDF:
Or explore our Application Notes pertaining to enzymes and other topics here
Table 8. Amplite® Enzyme Activity Kits For Assay Kits
In addition to the kits in the table above, AAT Bioquest offers a range of kits specifically for investigating the activity of caspases. Click the button below to learn more: Learn More
Luciferase Reporter Gene
Luciferase activity was measured with Amplite® Luciferase Reporter Gene Assay Kit
in a white 96-well plate with a NOVOstar plate reader (BMG Labtech). The luciferase activity is still detectable at 0.1 pg/well with 20 minutes incubation.
reporter gene, originally isolated from the common eastern firefly Photinus pyralis
, is the most popular choice for detection assays and other uses due to its low toxicity and ease of use. Our Amplite®
kits employ different variations of this useful gene, isolated from the firefly, sea pansy (Renilla reniformis
) and aquatic copepod Gaussia prince
for sensitive, efficient detection and steady signal. AAT Bioquest also offers its bioluminescent substrate Luciferin
in several forms.
Table 9. Amplite® Luciferase Reporter Gene Kits For Assay Kits
Horseradish Peroxidase (HRP)
The peroxidase enzyme family plays a prominent role in multiple cellular processes. They catalyze hydrogen peroxide (H2O2) oxidation of a variety of organic compounds, many of which are commonly present in living cells. For use in the laboratory, the most commonly used one is isolated from the common horseradish. Horseradish peroxidase (HRP) is a small, robust molecule of only 44kD, and can be easily conjugated to antibodies in a 4:1 ratio with little to no steric hindrance. Since H202 is a common product of many cellular pathways, the ability to quantify it makes HRP incredibly valuable for multiple biochemical applications. Learn More
Table 10. Amplite® HRP Assay Kits For Assay Kits
Alkaline Phosphatase (ALP)
Alkaline phosphatase (ALP) is an 86 kDa homodimeric protein enzyme present in multiple tissue types within the body, with the highest concentrations in the bone and liver. It is used as a biomarker for several pathologies. ALP is also a common reporter enzyme used in multiple immunoassays and other applications. It is typically conjugated to an antibody, and can be detected in multiple formats. The substrates for the Amplite®
ALP kits range from the popular para-Nitrophenylphosphate (pnPP) for colorimetric detection and 4-Methylumbelliferyl phosphate (MUP) for fluorimetric, to our proprietary substrate for luminometric platforms. Learn More
Table 11. Amplite® ALP Assay Kits For Assay Kits
The application of Signal Guard™ phosphatase reaction stopping solution in Amplite®
Alkaline Phosphatase Colorimetric Assay (AAT Bioquest Cat#11950
). Two parallel reactions containing 100 mU/mL Alkaline phosphatase were initiated by adding 200 ?L reaction mixture. The plots demonstrate that the reaction was completely inhibited by Signal Guard™ phosphatase reaction stopping solution.
Phosphatase Assays and Reaction Control
- The Signal Guard™ phosphatase reaction stopping solution is a ready to use reagent that provides a convenient tool for terminating fluorescence and colorimetric signal-generating phosphatase reactions at a user-determined time point, and also keep the fluorescence or colorimetric signal stable for up to 18 hours.
- The stopping solution is optimized and compatible with alkaline, acid and protein phosphatase.
- Compatible with nearly all colorimetric and fluorimetric phosphatase substrates, the solution requires only mild pH and only a 2-step protocol. Full details and pdf protocol available here.
Nicotinamide adenine dinucleotides are prolific cofactors that facilitate the reversible oxidation and reduction steps in all major metabolic pathways. They are present in varying concentrations in phosphorylated (NADP and NADPH) and unphosphorylated forms (NAD+ and NADH+). Since the NAD(P)(H) cofactors link cell metabolism to other systems in the body (including transcriptional control and cell signaling) they are a target for drug discovery and oncology research. By monitoring the changes in intracellular concentration of these biomolecules, researchers gain understanding of cell behavior and pathologies. Learn More
Table 12. Amplite® NAD/NADH Assay Kits For Assay Kits
Table 13. Amplite® NADP/NADPH Assay Kits For Assay Kits
Cellular Energy and Metabolism
All living cells must be able to fuel themselves with some form of energy. The process of transforming outside sources of energy (e.g. sunlight, food) into adenosine triphosphate (ATP) is the beginning of all life processes. Major metabolic pathways such as glycolysis are heavily regulated and include multiple enzymes and molecular intermediates. Assays to quantify changes in the concentrations of these biomolecules give valuable information about pathological mechanisms as well as normal cell activities.
Click the button on the right to view or download our PDF:
Or explore our digital catalog page for cell metabolism here
Table 14. Amplite® Metabolic Assay Kits For Assay Kits
Antioxidants and ROS
Reactive oxygen species (ROS)
are a major component of cell signaling
, gene expression, and other activities, with most occurring in the mitochondria
. The amount of ROS in the cell is usually at low concentrations, as they are used for multiple cell signaling activities and other purposes. The levels are also kept low by the presence of antioxidants, which are synthesized or imported by the cell. In high concentrations however, these chemically-reactive molecules can be very damaging and even lethal.
Being able to accurately measure ROS levels is a useful way to detect the early stages of multiple pathologies, as well as the activity of metabolites or normal/atypical cellular function.
Table 15. Amplite® ROS and Antioxidant Assay Kits For Assay Kits
In addition to the kits in the table above, AAT Bioquest offers a range of kits specifically for investigating the activity of Glutathione (GSH and GSSG)
. Click the button below to learn more: Learn More
As the building blocks of all proteins, amino acids play a role in every aspect of cellular activity. Sensitive, accurate measurements of these small compounds, both essential and non-essential, are relevant in multiple fields of study.
Table 16. Amplite® Amino Acid Assay Kits For Assay Kits
Table 17. Amplite® Kits For Human Serum Albumin Binding
Sphingomyelin and acetylcholine, along with other roles, are important to signal transduction in the central nervous system. Using our proprietary Amplite®
fluorescent substrates, these kits provide optimized protocols to give accurate, sensitive biomolecule quantification.
AAT Bioquest also offers Amplite®
assay kits for the detection of the enzymes associated with these biomolecules, namely acetylcholinesterase (AChE) and sphingomyelinase (SMase), in a several formats and colors. Acetylcholinesterase (AChE) and sphingomyelinase (SMase) assays are one of the most popular methods for investigating nerve response and function. For more in-depth information on these critical tests see below:
Table 18. Amplite® Neurotransmitter and Nerve Cell Signaling Assay Kits For Assay Kits
Specific biomolecules have distinctive changes in concentrations as a symptom of some pathologies. By measuring the changing levels of these materials against known standards, researchers gain insight into physiological conditions. Our Amplite®
kits provide sensitive quantification with a minimal sample size of only 100 µL reaction volume.
Table 19. Amplite® Biomarker Assay Kits For Asssay Kits