Human PKC zeta (Protein Kinase C Zeta) ELISA Kit (HUES02259)

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HUES02259
€539

Description

ELISA Kit Technical ManualMSDS

Human PKC zeta (Protein Kinase C Zeta) 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 Human PKC zeta. 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 PKC zeta 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 PKC zeta, 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 PKC zeta. The concentration of Human PKC zeta in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human PKC zeta in samples. No significant cross-reactivity or interference between Human PKC zeta 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
202.315
2.343
2.3292.271
101.497
1.515
1.5061.448
50.847
0.839
0.8430.785
2.50.426
0.43
0.4280.37
1.250.256
0.23
0.2430.185
0.630.165
0.135
0.150.092
0.310.099
0.111
0.1050.047
00.054
0.062
0.058--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.971.678.630.991.799.19
Standard deviation0.050.100.390.060.080.35
C V (%)5.155.994.526.064.473.81

Recovery

The recovery of Human PKC zeta 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)93-109100
EDTA plasma (n=5)84-9891
Cell culture media (n=5)88-10194

Linearity

Samples were spiked with high concentrations of Human PKC zeta 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 (%)84-9886-10093-109
Average (%)9192101
1:4Range (%)93-10682-9383-95
Average (%)1008889
1:8Range (%)89-10180-9189-102
Average (%)948695
1:16Range (%)90-10485-9886-102
Average (%)989293

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 ×12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100×)1 vial, 120 uL
Concentrated HRP Conjugate (100×)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 (25×)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

Human PKC zeta (Protein Kinase C Zeta) ELISA Kit (HUES02259) 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.

Human PKC zeta (Protein Kinase C Zeta) ELISA Kit (HUES02259) Protein Information

UniProt Protein Function:PKCZ: an AGC kinase of the PKC family. An atypical PKC: its activity is not regulated by Ca2+, PS, DAG or phorbol esters. Constitutively active.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 1p36.33-p36.2

Cellular Component: cell junction; cytoplasm; cytosol; intercellular junction; membrane; plasma membrane; vesicle

Molecular Function:insulin receptor substrate binding; protein binding; protein kinase activity; protein kinase C activity; protein serine/threonine kinase activity

Biological Process: activation of NF-kappaB transcription factor; establishment of cell polarity; negative regulation of insulin receptor signaling pathway; negative regulation of peptidyl-tyrosine phosphorylation; negative regulation of protein complex assembly; peptidyl-serine phosphorylation; positive regulation of insulin receptor signaling pathway; positive regulation of interleukin-4 production; positive regulation of T-helper 2 cell differentiation; protein amino acid phosphorylation; signal transduction; transforming growth factor beta receptor signaling pathway

NCBI Summary:Protein kinase C (PKC) zeta is a member of the PKC family of serine/threonine kinases which are involved in a variety of cellular processes such as proliferation, differentiation and secretion. Unlike the classical PKC isoenzymes which are calcium-dependent, PKC zeta exhibits a kinase activity which is independent of calcium and diacylglycerol but not of phosphatidylserine. Furthermore, it is insensitive to typical PKC inhibitors and cannot be activated by phorbol ester. Unlike the classical PKC isoenzymes, it has only a single zinc finger module. These structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from other isoenzymes of PKC. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:Q05513
NCBI GenInfo Identifier:68067736
NCBI Gene ID:5590
NCBI Accession:Q05513.4
UniProt Secondary Accession:Q05513,Q15207, Q5SYT5, Q969S4, A8K4N0, A8MU64, B7Z2J7 E9PCW2,
UniProt Related Accession:Q05513
Molecular Weight:56,136 Da
NCBI Full Name:Protein kinase C zeta type
NCBI Synonym Full Names:protein kinase C zeta
NCBI Official Symbol:PRKCZ  
NCBI Official Synonym Symbols:PKC2; PKC-ZETA  
NCBI Protein Information:protein kinase C zeta type
UniProt Protein Name:Protein kinase C zeta type
UniProt Synonym Protein Names:nPKC-zeta
Protein Family:Protein kinase
UniProt Gene Name:PRKCZ  
UniProt Entry Name:KPCZ_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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