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Cell Signaling

In biology, cell signaling is a network of communications that governs basic and complex cellular processes. Depending upon the nature of the signal, it can be classified as either biochemical or mechanical. Biochemical signals are generated by biochemical molecules such as proteins, lipids, ions or hormones. Detection of these biochemical agents may shed light on the state of that particular type of signaling pathway.

Disruption of signaling networks often leads to diseases such as cancer, immune diseases and metabolic disorders. By understanding signaling pathways, researchers and clinicians may be able to treat disease conditions effectively and efficiently. AAT Bioquest offers a broad range of assays to conduct cost-effective analysis of various enzymes, proteins or small molecules involved cell signaling pathways. Measure activities such as phosphorylation, methylation and acetylation with unparalleled sensitivity and reproducibility.



Adenosine Diphosphate Assays



ADP dose response was measured with the PhosphoWorks™ Fluorimetric ADP Assay Kit (Cat NO. 21655) in a solid black 384-well plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.3 µM ADP can be detected with 15, 30 minutes and 1 hour incubation (Z' factor =0.65).
Adenosine diphosphate (ADP) is an important organic molecule that plays a major role in cell metabolism. It is a product of ATP dephosphorylation, a key reaction in living cells that is essential for energy transfer, and can be recycled back to ATP via rephosphorylation. This interconversion of ADP and ATP occurs primarily in the mitochondria and chloroplasts by ATPases such as phosphatases, phosphorylases and kinases. ADP is stored in dense bodies inside blood platelets. When platelets are activated, ADP is released to interact with a family of purinergic receptors thereby regulating cellular function.

The PhosphoWorks™ Fluorimetric ADP assay offers a sensitive method for monitoring ADP formation. In this enzyme-coupled assay, ADP formation is measured by monitoring kinase activity due to their proportional relationship.

Table 1. Biochemical assays for measuring ADP activity, formation or depletion.

Assay
Ex (nm)
Em (nm)
Cutoff (nm)¹
Microplate Type
Unit Size
Cat No.
PhosphoWorks™ Fluorimetric ADP Assay Kit *Red Fluorescence*540590570Solid black100 tests21655
  1. Fluorescence microplate instrument specifications.

Adenosine Triphosphate Assays



ATP dose response was measured with the PhosphoWorks™ Luminescence ATP Assay Kit (Cat No. 21610) on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect 3 pmol ATP with 2 hours incubation (Z' factor = 0.6, blue 30 minutes, red 1 hour, and green 2 hours). The integration time was 1 sec. The half life is more than 1.5 hours.
Adenosine triphosphate (ATP) is a complex organic molecule that participates in cellular energenics, metabolic regulation and cellular signaling. It is present in all living organisms as the primary energy currency and rapidly degrades in the absence of viable organisms. As such, ATP determination is a useful tool utilized in a variety of research applications to identify the presence of viable organisms. Common applications include cell viability and cytotoxicity, detection of bacterial on surfaces, quantification of bacteria in water, somatic cells in culture, and in food quality and contamination tests.

Our PhosphoWorks™ Luminometric ATP assays provide a fast, simple and homogenous bioluminescence method that can be used for the determination of cell viability, cytotoxicity and proliferation in mammalian cells by ATP detection.

Table 2. Biochemical assays for measuring ATP activity, formation or depletion.

Assay
Ex/Abs (nm)
Em (nm)
Cutoff (nm)¹
Microplate Type
Unit Size
Cat No.
PhosphoWorks™ Fluorimetric ATP Assay Kit540590570Solid black100 tests21620
PhosphoWorks™ Colorimetric ATP Assay Kit570--Clear bottom100 tests21617
PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*---Solid white1 plate21610
PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*---Solid white10 plate21621
  1. Fluorescence microplate instrument specifications.

Cyclic Adenosine Monophosphate Assays



cAMP dose response was measured with Screen Quest™ Colorimetric ELISA cAMP Assay Kit (Cat#36370) in a clear 96-well plate with a SpectraMax microplate reader. The Absorbance can be read at 405 nm (blue line), 650 nm (red line) or 740 nm (Green line), the data in figure B are from the incubation with Amplite® Green for 3 hours.
Cyclic Adenosine Monophosphate (cAMP) is a vital second messenger utilized in many biological pathways. It is utilized for intracellular signal transduction in many organisms, conveying the cAMP-dependent pathway, and is involved in the activation of protein kinases and ion channel regulation. cAMP is derived from ATP and synthesized by plasma membrane-bound adenylyl cyclase which are coupled to transmembrane receptors. Activation of adenylyl cyclase is initiated by a range of signaling molecules. These molecules interact with receptors, such as GPCRs, which initiate adenylyl cyclase activity and increase intracellular cAMP levels. Finally, cAMP activates cAMP-dependent proteins kinases that are responsible for phosphorylating specific substrate proteins. Determination of cAMP activity is useful for functional assays assessing receptor activation, receptor characterization, and receptor-ligand identification.

Table 3. Biochemical assays for monitoring the activation of adenylyl cyclase in G-protein coupled receptor systems.

Assay
Ex/Abs (nm)
Em (nm)
Cutoff (nm)¹
Microplate Type
Unit Size
Cat No.
Screen Quest™ Fluorimetric ELISA cAMP Assay Kit540590570Solid black plate1 plates36373
Screen Quest™ Fluorimetric ELISA cAMP Assay Kit540590570Solid black plate10 plates36374
Screen Quest™ Colorimetric ELISA cAMP Assay Kit405, 650, 740--Clear plate1 plate36370
Screen Quest™ Colorimetric ELISA cAMP Assay Kit405, 650, 740--Clear plate10 plates36371
Screen Quest™ FRET No Wash cAMP Assay Kit---Solid black plate, black wall/clear bottom plate1 plates36379
Screen Quest™ FRET No Wash cAMP Assay Kit---Solid black plate, black wall/clear bottom plate10 plates36380
Screen Quest™ FRET No Wash cAMP Assay Kit---Solid black plate, black wall/clear bottom plate50 plates36381