Product Name:	Human PRKCG / PKC gamma ELISA Kit
Product Code:	HUFI02047
Size:	96 Assays
Alias:	PRKCG, PKCC, PKCG, SCA14, PKC-gamma, MGC57564, PKCCSCA14, PKCGprotein kinase C gamma type, protein kinase C, gamma
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PRKCG concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PRKCG and the recovery rates were calculated by comparing the measured value to the expected amount of Human PRKCG in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-105	98
EDTA plasma(n=5)	85-102	94
UFH plasma(n=5)	88-105	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PRKCG and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-103%	86-97%	87-105%
EDTA plasma(n=5)	84-97%	85-100%	85-97%
UFH plasma(n=5)	84-93%	80-99%	81-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P05129

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:Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; EC 2.7.11.13; Kinase, protein; AGC group; PKC family; Alpha subfamily Chromosomal Location of Human Ortholog: 19q13.4 Cellular Component: cytosol; dendrite; intracellular; perinuclear region of cytoplasm; plasma membrane Molecular Function:calcium-dependent protein kinase C activity; protein kinase activity; protein kinase C activity; protein serine/threonine/tyrosine kinase activity Biological Process: negative regulation of neuron apoptosis; negative regulation of protein catabolic process; negative regulation of protein ubiquitination; peptidyl-serine phosphorylation; phosphorylation; platelet activation; positive regulation of mismatch repair; protein amino acid phosphorylation; regulation of circadian rhythm; regulation of response to food; response to morphine; response to pain Disease: Spinocerebellar Ataxia 14
NCBI Summary:	Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play distinct roles in cells. The protein encoded by this gene is one of the PKC family members. This protein kinase is expressed solely in the brain and spinal cord and its localization is restricted to neurons. It has been demonstrated that several neuronal functions, including long term potentiation (LTP) and long term depression (LTD), specifically require this kinase. Knockout studies in mice also suggest that this kinase may be involved in neuropathic pain development. Defects in this protein have been associated with neurodegenerative disorder spinocerebellar ataxia-14 (SCA14). Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2015]
UniProt Code:	P05129
NCBI GenInfo Identifier:	462455
NCBI Gene ID:	5582
NCBI Accession:	P05129.3
UniProt Secondary Accession:	P05129,B7Z8Q0,
UniProt Related Accession:	P05129
Molecular Weight:	62,030 Da
NCBI Full Name:	Protein kinase C gamma type
NCBI Synonym Full Names:	protein kinase C gamma
NCBI Official Symbol:	PRKCG
NCBI Official Synonym Symbols:	PKCC; PKCG; SCA14; PKC-gamma
NCBI Protein Information:	protein kinase C gamma type
UniProt Protein Name:	Protein kinase C gamma type
Protein Family:	Protein kinase
UniProt Gene Name:	PRKCG
UniProt Entry Name:	KPCG_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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Human PRKCG / PKC gamma ELISA Kit

Description