Assay Kits

Inhibition of histone deacetylase (HDAC) has been implicated to modulate transcription and to induce apoptosis or differentiation in cancer cells. However, screening of compounds for HDAC inhibition has been difficult due to the lack of convenient tools for analyzing HDAC activity. The new HDAC Inhibitor Drug Screening Kit provides a fast, fluorescence-based method that eliminates radioactivity, extractions, or chromatography, as used in traditional assays. The new procedure requires only two easy steps, both performed on the same microtiter plate. First, your inhibitor candidates are mixed with HeLa Nuclear Extract and HDAC fluorometric substrate, which comprises an acetylated lysine side chain. Deacetylation of the substrate sensitizes the substrate, so that, in the second step, treatment with the Lysine Developer produces a fluorophore. The fluorophore can be easily analyzed using a fluorescence plate reader or a fluorometer. The assay is well suited for high throughput screening applications.

Histone deacetylases (HDACs) play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. HDAC1 is ubiquitously expressed and plays a role in cellular senescence, cardiomyocyte differentiation and hypertrophy, aging of the liver, myelination, and adult neurogenesis to name a few functions. The HDAC1 IP & Activity Assay Kit provides an antibody-based method to specifically immunoprecipitate the HDAC1 complex from cells & tissues and to measure HDAC1 activity fluorometrically. HDAC1 is immunoprecipitated from cell, nuclear or tissue extract(s) using HDAC1 antibody followed by capturing the complex on protein-A/G beads. The immunoprecipitated complex is incubated with the HDAC substrate. Only the deacetylated substrate is cleaved by the Developer to produce a fluorophore, which can be easily analyzed using a fluorescence plate reader.

Histone deacetylases (HDACs) play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. These proteins have crucial roles in development and physiology, especially in the heart and nervous system. It is also deeply involved in cellular proliferation, cell cycle and apoptosis. The HDAC2 IP & Activity Assay Kit provides an antibody-based method to specifically immunoprecipitate the HDAC2 complex from cells & tissues and to measure HDAC2 activity fluorometrically. HDAC2 is immunoprecipitated from cell or nuclear extract(s) using HDAC2 antibody followed by capturing the complex on protein-A/G beads. The immunoprecipitated complex is incubated with the HDAC substrate. Only the deacetylated substrate is cleaved by the Developer to produce a fluorophore, which can be easily analyzed using a fluorescence plate reader.

Histone deacetylases (HDACs) play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. These proteins have crucial roles in development and physiology, especially in the heart and nervous system. They are also deeply involved in cellular proliferation, cell cycle and apoptosis. HDAC3 is primarily localized in the nucleus, but can also be found in the cytoplasm and at the plasma membrane. The HDAC3 IP & Activity Assay Kit provides an antibody-based method to specifically immunoprecipitate the HDAC3 complex from cells & tissues and to measure HDAC3 activity fluorometrically. HDA3 is immunoprecipitated from cell or nuclear extract(s) using HDAC3 antibody followed by capturing the complex on protein-A/G beads. The immunoprecipitated complex is incubated with the HDAC substrate. Only the deacetylated substrate is cleaved by the Developer to produce a fluorophore, which can be easily analyzed using a fluorescence plate reader.

Histone deacetylases (HDACs) represent a large family of enzymes identified as key regulators of nucleosomal histone acetylation, a major event that controls eukaryotic gene transcription and are classified into three groups. They are believed to be involved in important biological activities, such as cell differentiation, proliferation, apoptosis, and senescence. In HDAC-3 Inhibitor screening Kit, HDAC-3 and developer will deacetylate and cleave the substrate [Arg-His-Lys-Lys(Ac)-AFC] to release the quenched fluorescent group (AFC), which can be detected at Em/Ex=380/500 nm. In presence of a HDAC-3 inhibitor, the cleavage will be inhibited. The kit provides a rapid, simple, sensitive, and reliable test, which is also suitable for high throughput screening of HDAC-3 inhibitors. Trichostatin A (TSA) is included as a control inhibitor to compare the efficacy of the test inhibitors.

Histone acetylases (HAT’s) and Histone deacetylases (HDAC’s) are associated with regulation of gene expression. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels of acetylation are associated with repression of gene expression. HDAC’s are localized in both the cytosol and nucleus and some shuttle between the nucleus and cytosol. Increased HDAC expression has been observed in various cancers. InSitu HDAC Activity Fluorometric Assay Kit provides a direct, fast, fluorescence-based method to measure the InSitu HDAC activity. The procedure requires two easy steps, all performed in the same cell culture plate. First, the cell permeable HDAC Substrate, which comprises an acetylated lysine side chain, is incubated with cells grown in a 96-well plate. Inside the cells, HDAC deacetylates the substrate. The second step involves lysing the cells and treating with the Developer that produces a fluorophore from the Deacetylated HDAC Substrate. The generated fluorescence can be quantified at Ex/Em = 368/442 nm. The assay is well suited for high throughput screening applications.

Irisin (human) Competitive ELISA kit is to be used for the in vitro quantitative determination of human irisin in cell culture supernatants, serum and plasma. It should also work for the in vitro quantitative determination of irisin in mouse, rat and monkey biological samples. A polyclonal antibody recognizing native irisin reacts with a series of predetermined recombinant irisin standard proteins or samples under competition in the irisin-coated plate. Their relative reactivity is plotted with that of the standard proteins. Detection limit: 1 ng/mL. Note: The Limit of detection was measured by adding two standard deviations to the mean value of 50 zero standard. Assay Range: 0.001 ug/mL – 5 ug/mL. Irisin levels range in human plasma and serum from 0.2 to > 2 ug /mL.

c-Jun N-terminal kinase (JNK) is one of the several main MAP kinase groups identified in mammals. Recent evidences suggest that activation of JNK may play an important role in neuronal apoptosis and other physiological and pathological processes. The JNK Activity Immunoassay Kit utilizes a JNK-specific antibody to immunoprecipitate JNK from cell lysate. Activity of the JNK is then determined in a kinase reaction using recombinant c-Jun as substrate. Phosphorylation of the c-Jun can be analyzed by Western blot analysis using a phospho-c-Jun specific antibody. The kit specifically detects JNK, other kinase activity would not be detected.

Lipid peroxidation is a type of oxidative damage that affects cellular membranes, lipoproteins, and other lipid-containing molecules. This process involves the breakdown of lipid peroxides, which are produced from polyunsaturated fatty acids, leading to the creation of malondialdehyde (MDA) and 4-hydroxyalkenals. Accurately measuring lipid peroxidation is crucial for evaluating oxidative stress under pathological conditions. Among the numerous methods, quantifying MDA, the final product of lipid peroxidation, is widely acknowledged as a reliable assay for oxidative damage assessment. Lipid Peroxidation Colorimetric Assay Kit offers a convenient means to detect MDA levels with sensitivity across a broad spectrum of substances including urine, serum, plasma, tissue extracts, cell lysate, cell culture media, and other biological fluids samples. In this assay, MDA in the sample reacts with thiobarbituric acid (TBA) to form an MDA-TBA adduct, which can be easily quantified using colorimetric techniques at a wavelength of 532 nm.

Lysosomes are membrane-bound organelles important for various cellular processes. They contain hydrolytic enzymes utilized in the metabolism of some biomolecules. The extracellular cargo (e.g. nutrients, toxins) binds to the cell membrane and is subsequently transported into membrane-bound endosomes for further degradation by lysosomes while intracellular components are transported to lysosomes through autophagy. Lysosomal dysfunction is associated with many human conditions such as aging and neurodegenerative disease. We have developed the Lysosomal Cytotoxicity Dual Staining Kit (cell-based) which contains two probes; membrane permeable, selective Lysosomal Stain and Cell Death Marker. We also include a Positive Control Reagent, which increases lysosome activity and staining, thus serves as an experimental control. This easy-to-use, non-radioactive kit allows studying the regulation of lysosome and cytotoxicity at the cellular level by using Fluorescence Microscopy and Flow Cytometry in cultured cells.

Malondialdehyde is a non-proteinogenic α-amino acid. It is a homologue of the amino acid cysteine, differing by an additional methylene bridge (-CH2-). Hypermalondialdehydemia has been correlated with the occurrence of blood clots, heart attacks and strokes, though it is unclear whether hypermalondialdehydemia is an independent risk factor for these conditions. It has also been associated with early pregnancy loss and with neural tube defects. Malondialdehyde ELISA kit is a competitive ELISA assay for the quantitative measurement of malondialdehyde in Human serum, plasma and cell culture supernatants. The density of color is proportional to the amount of malondialdehyde captured from the samples.

Disruption of the mitochondrial transmembrane potential is one of the earliest intracellular events that occur following induction of apoptosis. The MitoCapture Apoptosis Detection Kit provides a simple, fluorescent-based method for distinguishing between healthy and apoptotic cells by detecting the changes in the mitochondrial transmembrane potential. The kit utilizes MitoCapture, a cationic dye that fluoresces differently in healthy vs apoptotic cells. In healthy cells, MitoCapture accumulates and aggregates in the mitocondria, giving off a bright red fluorescence. In apoptotic cells, MitoCapture cannot aggregate in the mitochondria due to the altered mitochondrial transmembrane potential, and thus it remains in the cytoplasm in its monomer form, fluorescing green. The fluorescent signals can be easily detected by fluorescence microscopy using a band-pass filter (detects FITC and rhodamine) or analyzed by flow cytometry using FITC channel for green monomers (Ex/Em = 488/530+ 30 nm) and (optional) PI channel for red ggregates (Em = 488/590+ 42 nm).

Mitochondria are the power house of the cells and play an essential role in energy production. Damage to the mitochondria activates signaling pathways that induce apoptosis. Mitochondria also regulate and mediate transport of the metabolites and ions needed for oxidative phosphorylation and maintenance of membrane potential for ATP synthesis. Mitochondrial dysfunction leads to several disorders like cardiac dysfunction, diabetes, aging and neurological disorder, mainly caused by mutations in mitochondrial DNA or in nuclear genes that code for mitochondrial components. Thus, mitochondria have several different functions in the cell. CD Bioscience ready to use mitochondria Protein IP Buffer is optimized for immunoprecipitation (IP and co-IP) using mitochondria and mitochondrial extracts. The buffer is a gentle formulation, which maintains the stability of mitochondrial complexes. The Mitochondrial Protein IP kit is provided with different choices of detergents like n-Dodecyl-beta-D-maltoside, Triton X-100 and digitonin to achieve different stringency conditions for protein-protein interaction studies. Triton X-100 is the most commonly used detergent especially for membrane protein solubilization. However, in case of fragile complexes digitonin or n-Dodecyl-beta-D-maltoside is the choice of detergents.

Monoamine oxidases (MAO, EC 1.4.3.4) are a family of enzymes that can oxidize a wide variety of endogenous primary amines. Two isoforms, MAO-A and MAO-B, have been identified based on their substrate, inhibitor specificity, and tissue localization. MAO-A can oxidize primary amines such as serotonin and norepinephrine. MAO-A is a mitochondrial-bound enzyme that is ubiquitously expressed throughout the brain and other tissues. It has been implicated in panic, anxiety, and depression. Several MAO-A specific inhibitors such as clorgyline, brofaromine, toloxatone, tetrindole, etc. have been used as antidepressants, but their usage has been limited due to side effects. MAO-A Inhibitor Screening Kit offers a rapid, simple, sensitive, and reliable test suitable for high-throughput screening of MAO-A inhibitors. The assay is based on the fluorometric detection of H2O2, one of the byproducts generated during the oxidative deamination of MAO substrate.

Monoamine oxidases (MAO, EC 1.4.3.4) are a family of enzymes that can oxidize a wide variety of endogenous primary amines. Two isoforms, MAO-A and MAO-B, have been identified based on their substrate, inhibitor specificity, and tissue localization. MAO-B can oxidize primary amines, but its list of specific substrates (i.e. benzylamine, phenylethylamine) is more limited compared to MAO-A. MAO-B is a mitochondrial-bound enzyme that is ubiquitously expressed throughout the brain and other tissues. It has been investigated in numerous studies including Parkinson’s disease, Alzheimer’s, and tobacco addiction. Specific MAO-B inhibitors such as selegiline, & rasagiline have been used to treat Parkinson’s patients, but their benefits are considered rather modest. MAO-B Inhibitor Screening Kit offers a rapid, simple, sensitive, and reliable test suitable for high-throughput screening of MAO-B inhibitors. The assay is based on the fluorometric detection of H2O2, one of the byproducts generated during the oxidative deamination of MAO substrate.

Muellerian-inhibiting factor (AMH) is a glycoprotein, produced by the Sertoli cells of the testis, causes regression of the Muellerian duct. It is also able to inhibit the growth of tumors derived from tissues of Muellerian duct origin. Although it does not compete with EGF for receptor binding sites, MIS can inhibit the autophosphorylation of the EGF receptor in vitro. AMH ELISA kit is a sandwich ELISA assay for the quantitative measurement of AMH in Human serum, plasma and cell culture supernatants. The density of color is proportional to the amount of AMH captured from the samples.

Myeloperoxidase (MPO) is a peroxidase enzyme (EC 1.11.1.7) most abundantly present in neutrophil granulocytes. It is a green hemoprotein found in neutrophils and monocytes that catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates that play a role in the oxygen-dependent killing of tumor cells and microorganisms. Its heme pigment causes the green color in secretions rich in neutrophils, such as pus and some forms of mucus. Furthermore, it can oxidize tyrosine to a tyrosyl radical using hydrogen peroxide as an oxidizing agent. In MPO Assay Kit, MPO catalyzes the production of NaClO from H₂O₂ and NaCl. Subsequently, NaClO will react stoichiometrically with Aminophenyl fluorescein (APF) to generate fluorescein, which has a strong fluorescence and can be detected at Ex/Em = 485/525 nm. The kit provides a rapid, simple, sensitive, and reliable test suitable as a high throughput assay of MPO activity. This kit can be used to detect MPO activity as low as 0.5 μU per well.

NRG-1 (human) ELISA Kit is based on the standard principle of a sandwich enzyme-linked immunosorbent assay. A mouse monoclonal antibody specific for human NRG-1 is coated on a 96-well plate. Standards and test samples are added to the wells and NRG-1 present in a sample is bound by the immobilized antibody. A biotinylated polyclonal detection antibody from goat specific for NRG-1 is added subsequently. After washing away the unbound biotinylated antibody with PBS or TBS buffer, HRP conjugate is added to the wells. The wells are again washed with PBS or TBS buffer to remove the unbound conjugates. HRP substrate TMB is used to visualize the HRP enzymatic reaction. TMB is used by HRP to produce a blue product that changes into yellow after adding acidic stop solution. The density of yellow color is proportional to the human NRG-1 captured onto the plate. This ELISA kit shows no detectable cross-reactivity with related proteins. Detection Range: 62.5 pg/mL – 4000 pg/mL. Sensitivity: < 10 pg/mL. Coefficient of variation: 5% (Intra-assay) and 7% (Inter-assay).

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. NFKB1 ELISA kit is a sandwich ELISA assay for the quantitative measurement of human NFKB1 in serum, plasma and cell culture supernatants. The density of color is proportional to the amount of NFKB1 captured from the samples.

Retinol binding protein (RBP) 4 is the only specific transport protein for vitamin A in the circulation whose function is to deliver vitamin to target tissues. In obesity and type 2 diabetes, the expression of Glut4 is significantly impaired in adipocytes. Glucose transport via Glut4 is the rate-limiting step for glucose use by muscle and adipose tissue. Adipocyte-specific deletion of Gluts leads to notable elevation of mouse RBP4 causing systemic insulin resistance, and that reduction of RBP4 improves insulin resistance. This identified a novel role of RBP4 in regulating insulin action and RBP4 is recorded as an adipocyte-derived hormone. The RBP4 (human) ELISA Kit is to be used for the in vitro quantitative determination of human RBP4 in serum, urine and cell culture supernatant. This assay is a competitive ELISA which utilizies a 96-well microtiter plate which was pre-coated with a human RBP4. A purified polyclonal which recognizing native human RBP4 reacts with a series of predetermined recombinant human RBP4 standard proteins or the test samples under competition in the human RBP4-coated plate. Their relative reactivity is plotted with that of the standard proteins. This ELISA is specific for the measurement of natural and recombinant human RBP4. It does not cross-react with mouse RBP4, rat RBP4, human adiponectin, rat adiponectin, human resistin, human vaspin, human clusterin, human leptin, human IL-23, human IL-33, human GPX3, human Nampt, human ANG1, human ANG2, human ANGPTL3, human ANGPTL4, human ANGPTL6, human FABP4, human RELM-β, rat RELM-α, mouse Nampt, human PAI-1. The assay range is 0.001 – 5 µg RBP4/mL and a detection limit of 1 ng/mL (based on adding two standard deviations to the mean value of the (50) zero standards).