Human PKD1 (Protein Kinase D1) ELISA Kit (HUES02262)
- 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 Human PKD1 in samples. No significant cross-reactivity or interference between Human 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 Human 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 Human 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 Human 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 Human PKD1. The concentration of Human 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:EC 2. 7. 11. 13; Protein kinase, CAMK; Motility/polarity/chemotaxis; Autophagy; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; CAMK group; PKD family
Chromosomal Location of Human Ortholog: 14q11
Cellular Component: cytoplasm; cytosol; Golgi apparatus; integral to plasma membrane; plasma membrane; trans-Golgi network
Molecular Function:identical protein binding; kinase activity; protein binding; protein kinase C activity; protein serine/threonine kinase activity
Biological Process: activation of CREB transcription factor; activation of NF-kappaB transcription factor; cell proliferation; Golgi organization and biogenesis; Golgi vesicle transport; integrin-mediated signaling pathway; negative regulation of endocytosis; peptidyl-serine phosphorylation; positive regulation of angiogenesis; positive regulation of blood vessel endothelial cell migration; positive regulation of endothelial cell proliferation; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of osteoblast differentiation; positive regulation of peptidyl-serine phosphorylation; positive regulation of transcription from RNA polymerase II promoter; protein amino acid autophosphorylation; protein amino acid phosphorylation; Ras protein signal transduction; regulation of protein stability; signal transduction; sphingolipid biosynthetic process; vascular endothelial growth factor receptor signaling pathway
|NCBI Summary:||PRKD1 is a serine/threonine kinase that regulates a variety of cellular functions, including membrane receptor signaling, transport at the Golgi, protection from oxidative stress at the mitochondria, gene transcription, and regulation of cell shape, motility, and adhesion (summary by Eiseler et al. , 2009 [PubMed 19329994]). [supplied by OMIM, Nov 2010]|
|NCBI GenInfo Identifier:||209572639|
|NCBI Gene ID:||5587|
|NCBI Accession:||Q15139. 2|
|UniProt Secondary Accession:||Q15139,A6NL64, B2RAF6,|
|UniProt Related Accession:||Q15139|
|Molecular Weight:||101,704 Da|
|NCBI Full Name:||Serine/threonine-protein kinase D1|
|NCBI Synonym Full Names:||protein kinase D1|
|NCBI Official Symbol:||PRKD1|
|NCBI Official Synonym Symbols:||PKD; PKCM; 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|
|Protein Family:||Serine/threonine-protein kinase|
|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 Human PKD1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PKD1 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.35||5.56||4.57||6.54||4.17||3.44|
The recovery of Human 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)||92-106||99|
|Cell culture media (n=5)||89-101||95|
Samples were spiked with high concentrations of Human 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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.