Rat PKD2 (Protein Kinase D2) ELISA Kit (RTES00684)

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SKU:
RTES00684
€649

Description

ELISA Kit Technical ManualMSDS

Rat PKD2 (Protein Kinase D2) ELISA Kit

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 PKD2 . 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 PKD2 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 PKD2, 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 PKD2. The concentration of Rat PKD2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection MethodColormetric
Detection Range1.56-100 ng/mL
Sensitivity0.94 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat PKD2 in samples. No significant cross-reactivity or interference between Rat PKD2 and analogues was observed.

Typical Data

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
1002.335
2.341
2.3382.267
501.476
1.52
1.4981.427
250.898
0.864
0.8810.81
12.50.423
0.457
0.440.369
6.250.259
0.243
0.2510.18
3.130.168
0.16
0.1640.093
1.560.118
0.12
0.1190.048
00.061
0.081
0.071--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat PKD2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat PKD2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)5.1015.1546.845.2615.1245.48
Standard deviation0.290.631.520.350.831.57
C V (%)5.694.163.256.655.493.45

Recovery

The recovery of Rat PKD2 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)93-10799
EDTA plasma (n=5)93-109100
Cell culture media (n=5)91-10698

Linearity

Samples were spiked with high concentrations of Rat PKD2 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)
1:2Range (%)85-9997-11197-109
Average (%)91103103
1:4Range (%)95-10882-9682-93
Average (%)1008888
1:8Range (%)86-10083-9787-102
Average (%)939094
1:16Range (%)93-10581-9283-96
Average (%)1008689

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat PKD2 (Protein Kinase D2) ELISA Kit (RTES00684) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Rat PKD2 (Protein Kinase D2) ELISA Kit (RTES00684) Protein Information

UniProt Protein Function:Serine/threonine-protein kinase that converts transient diacylglycerol (DAG) signals into prolonged physiological effects downstream of PKC, and is involved in the regulation of cell proliferation via MAPK1/3 (ERK1/2) signaling, oxidative stress-induced NF-kappa-B activation, inhibition of HDAC7 transcriptional repression, signaling downstream of T-cell antigen receptor (TCR) and cytokine production, and plays a role in Golgi membrane trafficking, angiogenesis, secretory granule release and cell adhesion. May potentiate mitogenesis induced by the neuropeptide bombesin by mediating an increase in the duration of MAPK1/3 (ERK1/2) signaling, which leads to accumulation of immediate-early gene products including FOS that stimulate cell cycle progression. In response to oxidative stress, is phosphorylated at Tyr-438 and Tyr-718 by ABL1, which leads to the activation of PRKD2 without increasing its catalytic activity, and mediates activation of NF-kappa-B. In response to the activation of the gastrin receptor CCKBR, is phosphorylated at Ser-244 by CSNK1D and CSNK1E, translocates to the nucleus, phosphorylates HDAC7, leading to nuclear export of HDAC7 and inhibition of HDAC7 transcriptional repression of NR4A1/NUR77. Upon TCR stimulation, is activated independently of ZAP70, translocates from the cytoplasm to the nucleus and is required for interleukin-2 (IL2) promoter up-regulation. During adaptive immune responses, is required in peripheral T-lymphocytes for the production of the effector cytokines IL2 and IFNG after TCR engagement and for optimal induction of antibody responses to antigens. In epithelial cells stimulated with lysophosphatidic acid (LPA), is activated through a PKC-dependent pathway and mediates LPA-stimulated interleukin-8 (IL8) secretion via a NF-kappa-B-dependent pathway. During TCR-induced T-cell activation, interacts with and is activated by the tyrosine kinase LCK, which results in the activation of the NFAT transcription factors. In the trans-Golgi network (TGN), regulates the fission of transport vesicles that are on their way to the plasma membrane and in polarized cells is involved in the transport of proteins from the TGN to the basolateral membrane. Plays an important role in endothelial cell proliferation and migration prior to angiogenesis, partly through modulation of the expression of KDR/VEGFR2 and FGFR1, two key growth factor receptors involved in angiogenesis. In secretory pathway, is required for the release of chromogranin-A (CHGA)-containing secretory granules from the TGN. Downstream of PRKCA, plays important roles in angiotensin-2-induced monocyte adhesion to endothelial cells.
UniProt Code:Q5XIS9
NCBI GenInfo Identifier:62078527
NCBI Gene ID:292658
NCBI Accession:NP_001013917.1
UniProt Related Accession:Q5XIS9
Molecular Weight:96,497 Da
NCBI Full Name:serine/threonine-protein kinase D2
NCBI Synonym Full Names:protein kinase D2
NCBI Official Symbol:Prkd2  
NCBI Official Synonym Symbols:RGD1308054  
NCBI Protein Information:serine/threonine-protein kinase D2
UniProt Protein Name:Serine/threonine-protein kinase D2
UniProt Synonym Protein Names:nPKC-D2
Protein Family:Serine/threonine-protein kinase
UniProt Gene Name:Prkd2  
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Additional Information

Reactivity:
Rat
ELISA Type:
Sandwich
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