Description
Human PDE4D (Phosphodiesterase 4D, cAMP Specific) ELISA Kit
The Human PDE4D (Phosphodiesterase 4D) cAMP-Specific ELISA Kit is specifically designed for the accurate measurement of PDE4D levels in human samples including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.PDE4D is an important enzyme involved in the regulation of cyclic AMP (cAMP) levels in cells, playing a crucial role in various physiological processes including cell signaling and gene expression.
Dysregulation of PDE4D has been linked to numerous diseases such as inflammatory disorders, neurodegenerative diseases, and cancer, highlighting its significance as a potential therapeutic target and biomarker for disease progression.By utilizing the Human PDE4D cAMP-Specific ELISA Kit, researchers can accurately assess PDE4D levels in human samples, furthering our understanding of its role in physiological and pathological conditions, and aiding in the development of novel treatment strategies.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human PDE4D in samples. No significant cross-reactivity or interference between Human PDE4D and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human PDE4D. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human PDE4D and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human PDE4D, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human PDE4D. The concentration of Human PDE4D in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | PDE4D: Hydrolyzes the second messenger cAMP, which is a key regulator of many important physiological processes. Homodimer for the long isoforms. Isoforms with truncated N-termini are monomeric. Isoform 3 is part of a ternary complex containing PRKAR2A, PRKAR2B and AKAP9. Interacts with PDE4DIP. Identified in a complex composed of RYR1, PDE4D, PKA, FKBP1A and protein phosphatase 1 (PP1). Isoform 5, isoform N3 and isoform 12 bind GNB2L1 via their unique N-terminus. Binds ARRB2. Widespread; most abundant in skeletal muscle. Isoform 6 is detected in brain. Isoform 8 is detected in brain, placenta, lung and kidney. Isoform 7 is detected in heart and skeletal muscle. Inhibited by rolipram. Activated by phosphatidic acid. Belongs to the cyclic nucleotide phosphodiesterase family. PDE4 subfamily. 12 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:EC 3. 1. 4. 53; Nucleotide Metabolism - purine; Phosphodiesterase Chromosomal Location of Human Ortholog: 5q12 Cellular Component: Golgi apparatus; centrosome; myofibril; membrane; apical plasma membrane; voltage-gated calcium channel complex; cytosol Molecular Function:3',5'-cyclic-AMP phosphodiesterase activity; protein binding; enzyme binding; metal ion binding; ubiquitin protein ligase binding; beta-2 adrenergic receptor binding; drug binding; cAMP binding; SH3 domain binding; 3',5'-cyclic-nucleotide phosphodiesterase activity; ATPase binding Biological Process: neutrophil chemotaxis; negative regulation of heart contraction; regulation of heart rate; positive regulation of smooth muscle cell proliferation; multicellular organism growth; cAMP catabolic process; negative regulation of peptidyl-serine phosphorylation; positive regulation of smooth muscle cell migration; T cell receptor signaling pathway; memory; positive regulation of interferon-gamma production; cAMP-mediated signaling; cellular protein complex assembly; smooth muscle contraction; positive regulation of interleukin-2 production; regulation of receptor activity; positive regulation of interleukin-5 production; regulation of G-protein coupled receptor protein signaling pathway; lung development; aging |
| NCBI Summary: | This gene encodes one of four mammalian counterparts to the fruit fly 'dunce' gene. The encoded protein has 3',5'-cyclic-AMP phosphodiesterase activity and degrades cAMP, which acts as a signal transduction molecule in multiple cell types. This gene uses different promoters to generate multiple alternatively spliced transcript variants that encode functional proteins. [provided by RefSeq, Sep 2009] |
| UniProt Code: | Q08499 |
| NCBI GenInfo Identifier: | 12644392 |
| NCBI Gene ID: | 5144 |
| NCBI Accession: | Q08499. 2 |
| UniProt Secondary Accession: | Q08499,O43433, Q13549, Q13550, Q13551, Q7Z2L8, Q8IV84 Q8IVA9, Q8IVD2, Q8IVD3, Q96HL4, Q9HCX7, |
| UniProt Related Accession: | Q08499 |
| Molecular Weight: | 809 |
| NCBI Full Name: | cAMP-specific 3',5'-cyclic phosphodiesterase 4D |
| NCBI Synonym Full Names: | phosphodiesterase 4D, cAMP-specific |
| NCBI Official Symbol: | PDE4D |
| NCBI Official Synonym Symbols: | DPDE3; PDE43; STRK1; ACRDYS2; HSPDE4D; PDE4DN2 |
| NCBI Protein Information: | cAMP-specific 3',5'-cyclic phosphodiesterase 4D; cAMP-specific phosphodiesterase PDE4D6; phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog, Drosophila) |
| UniProt Protein Name: | cAMP-specific 3',5'-cyclic phosphodiesterase 4D |
| UniProt Synonym Protein Names: | DPDE3; PDE43 |
| Protein Family: | cAMP-specific 3',5'-cyclic phosphodiesterase |
| UniProt Gene Name: | PDE4D |
| UniProt Entry Name: | PDE4D_HUMAN |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
| 20 | 2.495 2.509 | 2.502 | 2.424 |
| 10 | 1.67 1.68 | 1.675 | 1.597 |
| 5 | 0.961 0.949 | 0.955 | 0.877 |
| 2.5 | 0.496 0.52 | 0.508 | 0.43 |
| 1.25 | 0.309 0.285 | 0.297 | 0.219 |
| 0.63 | 0.19 0.176 | 0.183 | 0.105 |
| 0.31 | 0.128 0.134 | 0.131 | 0.053 |
| 0 | 0.074 0.082 | 0.078 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PDE4D were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PDE4D were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 0.97 | 3.12 | 8.53 | 0.94 | 2.93 | 7.99 |
| Standard deviation | 0.05 | 0.15 | 0.40 | 0.06 | 0.15 | 0.37 |
| C V (%) | 5.15 | 4.81 | 4.69 | 6.38 | 5.12 | 4.63 |
Recovery
The recovery of Human PDE4D spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 86-100 | 93 |
| EDTA plasma (n=5) | 90-106 | 97 |
| Cell culture media (n=5) | 96-106 | 101 |
Linearity
Samples were spiked with high concentrations of Human PDE4D and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 96-112 | 89-102 | 92-108 |
| Average (%) | 104 | 96 | 99 | |
| 1:4 | Range (%) | 94-106 | 85-96 | 84-97 |
| Average (%) | 99 | 91 | 91 | |
| 1:8 | Range (%) | 86-98 | 84-96 | 88-98 |
| Average (%) | 92 | 91 | 93 | |
| 1:16 | Range (%) | 92-103 | 82-93 | 86-101 |
| Average (%) | 98 | 88 | 93 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
- Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
