Rat PKCG (Protein Kinase C Gamma) ELISA Kit (RTES00678)

(No reviews yet) Write a Review
SKU:
RTES00678
€649

Description

ELISA Kit Technical ManualMSDS

Rat PKCG (Protein Kinase C Gamma) 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 PKCG . 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 PKCG 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 PKCG, 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 PKCG. The concentration of Rat PKCG 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 Range3.13-200 ng/mL
Sensitivity1.88 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Rat PKCG in samples. No significant cross-reactivity or interference between Rat PKCG 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
2002.344
2.362
2.3532.283
1001.546
1.598
1.5721.502
500.856
0.854
0.8550.785
250.473
0.501
0.4870.417
12.50.232
0.214
0.2230.153
6.250.177
0.155
0.1660.096
3.130.112
0.128
0.120.05
00.069
0.071
0.07--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)9.8225.5590.3010.2226.5092.34
Standard deviation0.571.223.210.661.184.95
C V (%)5.804.773.556.464.455.36

Recovery

The recovery of Rat PKCG 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)89-10095
EDTA plasma (n=5)87-10093
Cell culture media (n=5)93-108100

Linearity

Samples were spiked with high concentrations of Rat PKCG 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 (%)92-10389-10190-103
Average (%)979697
1:4Range (%)93-10585-9686-98
Average (%)1009192
1:8Range (%)93-10782-9383-96
Average (%)988689
1:16Range (%)93-10983-9787-102
Average (%)998994

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 PKCG (Protein Kinase C Gamma) ELISA Kit (RTES00678) 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 PKCG (Protein Kinase C Gamma) ELISA Kit (RTES00678) Protein Information

UniProt Protein Function:PKCG: a calcium-activated, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase. Expressed in the brain and spinal cord where its localization is restricted to neurons. Several neuronal functions, including long term potentiation and depression (LTP<D) specifically require this kinase. Knockout studies in mice also suggest that this kinase may be involved in neuropathic pain development. Defects have been associated with neurodegenerative disorder spinocerebellar ataxia-14. Plays diverse roles in neuronal cells and eye tissues, such as regulation of the neuronal receptors GLUR4 and NMDAR1, modulation of receptors and neuronal functions related to sensitivity to opiates, pain and alcohol, mediation of synaptic function and cell survival after ischemia, and inhibition of gap junction activity after oxidative stress. Binds and phosphorylates GLUR4 glutamate receptor and regulates its function by increasing plasma membrane-associated GRIA4 expression. In primary cerebellar neurons treated with the agonist 3,5-dihyidroxyphenylglycine, functions downstream of the metabotropic glutamate receptor MGLUR5 and phosphorylates NMDAR1 receptor which plays a key role in synaptic plasticity, synaptogenesis, excitotoxicity, memory acquisition and learning. May be involved in the regulation of hippocampal long-term potentiation (LTP), but may be not necessary for the process of synaptic plasticity. May modulate the functionality of mu-type-opioid receptors by participating in a signaling pathway which leads to the phosphorylation and degradation of opioid receptors. May also contributes to chronic morphine-induced changes in nociceptive processing. Plays a role in neuropathic pain mechanisms and contributes to the maintenance of the allodynia pain produced by peripheral inflammation. Plays an important role in initial sensitivity and tolerance to ethanol, by mediating the behavioral effects of ethanol as well as the effects of this drug on the GABA(A) receptors. During and after cerebral ischemia modulate neurotransmission and cell survival in synaptic membranes, and is involved in insulin-induced inhibition of necrosis, an important mechanism for minimizing ischemic injury. Required for the elimination of multiple climbing fibers during innervation of Purkinje cells in developing cerebellum. Is activated in lens epithelial cells upon hydrogen peroxide treatment, and phosphorylates connexin-43, resulting in disassembly of GJA1 gap junction plaques and inhibition of gap junction activity which could provide a protective effect against oxidative stress. Phosphorylates p53 and promotes p53-dependent apoptosis in response to DNA damage. Interacts with GRIA4. Interacts with CDCP1. Interacts with TP53INP1 and p53. Expressed in Purkinje cells of the cerebellar cortex.
UniProt Protein Details:

Protein type:Kinase, protein; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.13; Protein kinase, AGC; AGC group; PKC family; Alpha subfamily

Cellular Component: neuron projection; perinuclear region of cytoplasm; cytoplasm; dendrite; plasma membrane; intercellular junction; cytosol; nucleus

Molecular Function:protein binding; protein kinase C activity; zinc ion binding; protein serine/threonine/tyrosine kinase activity; calcium-dependent protein kinase C activity; ATP binding; protein kinase activity

Biological Process: synaptic transmission; positive regulation of mismatch repair; learning and/or memory; protein amino acid autophosphorylation; rhythmic process; response to morphine; chemosensory behavior; negative regulation of protein ubiquitination; response to pain; negative regulation of neuron apoptosis; negative regulation of protein catabolic process; regulation of circadian rhythm; phosphorylation; regulation of response to food

NCBI Summary:enriched in the neurons of the central nervous system and the spinal cord [RGD, Feb 2006]
UniProt Code:P63319
NCBI GenInfo Identifier:6981400
NCBI Gene ID:24681
NCBI Accession:NP_036760.1
UniProt Secondary Accession:P63319,P05697, Q5FWS3,
UniProt Related Accession:P63319
Molecular Weight:
NCBI Full Name:protein kinase C gamma type
NCBI Synonym Full Names:protein kinase C, gamma
NCBI Official Symbol:Prkcg  
NCBI Official Synonym Symbols:PKC; PKCI; Prkc; Prkcc; RATPKCI  
NCBI Protein Information:protein kinase C gamma type; PKC-gamma; protein kinase C type I (gamma type)
UniProt Protein Name:Protein kinase C gamma type
UniProt Gene Name:Prkcg  
UniProt Entry Name:KPCG_RAT
View AllClose

Additional Information

Reactivity:
Rat
ELISA Type:
Sandwich
View AllClose