Rat PKD1 (Protein Kinase D1) ELISA Kit (RTES00683)
- Product type:
- ELISA Kit
- ELISA Type:
|Detection Range:||1.56-100 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Rat PKD1 in samples. No significant cross-reactivity or interference between Rat PKD1 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 Rat PKD1. 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 Rat PKD1 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 Rat PKD1, 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 Rat PKD1. The concentration of Rat PKD1 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||PRKD1: a CAMK kinase of the PKD family. Cleavage by caspase-3 following DNA damage activates it and alters its subcellular localization. Sensitizes cells to apoptosis induced by genotoxic stress. Its cleavage is blocked in cells that over-express the anti-apoptotic Bcl-x(L) protein. Expression of a caspase-resistant mutant partially inhibits DNA damage-induced apoptosis. Its activation by TLR ligands is dependent on MyD88, IRAK4 and -1, but not TRAF6. Essential for MyD88-dependent proinflammatory immune responses. Activated by diacylglycerol and phorbol esters. Binds to the trans-Golgi network and regulates the fission of transport carriers specifically destined to the cell surface. Colocalizes with F-actin at peripheral F-actin-rich structures in membrane ruffles at the edge of lamellipodia in cervical carcinoma cells. Substrates reportedly include critical regulatory proteins including CREB, SSH1L, CTNNB1, HDACs 5 and 7, PKD1, HPK1, MARK2, PIP5K2A, PPP1R14A|
|UniProt Protein Details:|
Protein type:Kinase, protein; Autophagy; Protein kinase, CAMK; Protein kinase, Ser/Thr (non-receptor); Motility/polarity/chemotaxis; EC 2. 7. 11. 13; CAMK group; PKD family
Chromosomal Location of Human Ortholog: 14q11
Cellular Component: integral to plasma membrane; cytoplasm; plasma membrane; intercellular junction; trans-Golgi network; cell cortex; nucleus; cytosol
Molecular Function:identical protein binding; protein serine/threonine kinase activity; protein binding; protein kinase C activity; metal ion binding; ATP binding
Biological Process: integrin-mediated signaling pathway; sphingolipid metabolic process; positive regulation of I-kappaB kinase/NF-kappaB cascade; sphingolipid biosynthetic process; apoptosis; protein amino acid autophosphorylation; negative regulation of endocytosis; positive regulation of blood vessel endothelial cell migration; activation of CREB transcription factor; signal transduction; positive regulation of peptidyl-serine phosphorylation; activation of NF-kappaB transcription factor; Golgi vesicle transport; cell proliferation; positive regulation of osteoblast differentiation; peptidyl-serine phosphorylation; positive regulation of angiogenesis; innate immune response; positive regulation of transcription from RNA polymerase II promoter; positive regulation of endothelial cell proliferation; angiogenesis; vascular endothelial growth factor receptor signaling pathway; inflammatory response; Golgi organization and biogenesis
|NCBI Summary:||The protein encoded by this gene is a serine/threonine protein kinase involved in many cellular processes, including Golgi body membrane integrity and transport, cell migration and differentiation, MAPK8/JNK1 and Ras pathway signaling, MAPK1/3 (ERK1/2) pathway signaling, cell survival, and regulation of cell shape and adhesion. [provided by RefSeq, Jan 2017]|
|NCBI GenInfo Identifier:||115529463|
|NCBI Gene ID:||5587|
|NCBI Accession:||NP_002733. 2|
|UniProt Secondary Accession:||Q9WTQ1,Q62101, Q9WTQ1,|
|UniProt Related Accession:||Q15139|
|NCBI Full Name:||serine/threonine-protein kinase D1 isoform 2|
|NCBI Synonym Full Names:||protein kinase D1|
|NCBI Official Symbol:||PRKD1|
|NCBI Official Synonym Symbols:||PKD; PKCM; CHDED; PRKCM; PKC-MU|
|NCBI Protein Information:||serine/threonine-protein kinase D1|
|UniProt Protein Name:||Serine/threonine-protein kinase D1|
|UniProt Synonym Protein Names:||Protein kinase C mu type; Protein kinase D; nPKC-D1; nPKC-mu|
|UniProt Gene Name:||PRKD1|
|UniProt Entry Name:||KPCD1_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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat PKD1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat PKD1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.88||4.64||3.65||6.20||4.16||5.19|
The recovery of Rat PKD1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||92-102||97|
|Cell culture media (n=5)||91-107||97|
Samples were spiked with high concentrations of Rat PKD1 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)|
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.
|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.