Product Name:	Human PKC alpha / PRKCA ELISA Kit
Product Code:	HUFI01258
Size:	96 Assays
Alias:	PRKCA, Protein kinase C alpha type, PKCA, AAG6, PKC-A, PKC-alpha, PRKACA, aging-associated gene 6, protein kinase C, alpha
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PRKCA 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 PRKCA and the recovery rates were calculated by comparing the measured value to the expected amount of Human PRKCA in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-104	99
EDTA plasma(n=5)	92-103	97
UFH plasma(n=5)	85-102	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PRKCA 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)	86-105%	86-97%	87-94%
EDTA plasma(n=5)	83-92%	83-97%	83-89%
UFH plasma(n=5)	81-90%	81-98%	80-97%

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

P17252

UniProt Protein Function:	PKCA: an AGC kinase of the PKC family. A classical PKC downstream of many mitogenic and receptors. Classical PKCs are calcium-dependent enzymes that are activated by phosphatidylserine, diacylglycerol and phorbol esters. Contains a pseudo-substrate autoinhibitory domain that binds to the catalytic domain preventing its activation in the absence of cofactors or activators.
UniProt Protein Details:	Protein type:EC 2.7.11.13; Protein kinase, Ser/Thr (non-receptor); Oncoprotein; Kinase, protein; Protein kinase, AGC; AGC group; PKC family; Alpha subfamily Chromosomal Location of Human Ortholog: 17q22-q23.2 Cellular Component: nucleoplasm; photoreceptor outer segment; mitochondrion; cell soma; endoplasmic reticulum; perinuclear region of cytoplasm; apical part of cell; mitochondrial membrane; dendrite; cytoplasm; plasma membrane; cytosol Molecular Function:protein serine/threonine kinase activity; protein binding; enzyme binding; protein kinase C activity; zinc ion binding; calcium-dependent protein kinase C activity; ATP binding; protein kinase activity Biological Process: phototransduction, visible light; extracellular matrix organization and biogenesis; positive regulation of cell adhesion; nerve growth factor receptor signaling pathway; regulation of muscle contraction; mitotic nuclear envelope disassembly; positive regulation of lipopolysaccharide-mediated signaling pathway; negative regulation of insulin receptor signaling pathway; signal transduction; positive regulation of mitotic cell cycle; protein amino acid phosphorylation; induction of positive chemotaxis; synaptic transmission; negative regulation of cell proliferation; chondrocyte differentiation; angiogenesis; cell adhesion; positive regulation of macrophage differentiation; regulation of peptidyl-tyrosine phosphorylation; regulation of the force of heart contraction; inactivation of MAPK activity; epidermal growth factor receptor signaling pathway; platelet activation; neutrophil chemotaxis; fibroblast growth factor receptor signaling pathway; positive regulation of blood vessel endothelial cell migration; adenylate cyclase activation; negative regulation of adenylate cyclase activity; cellular calcium ion homeostasis; rhodopsin mediated signaling; negative regulation of glucose import; positive regulation of angiogenesis; regulation of rhodopsin mediated signaling; phospholipase C activation; energy reserve metabolic process; innate immune response; gene expression; positive regulation of endothelial cell proliferation; positive regulation of protein amino acid phosphorylation; mitotic cell cycle; blood coagulation; vascular endothelial growth factor receptor signaling pathway; regulation of insulin secretion; positive regulation of cell migration; positive regulation of inflammatory response
NCBI Summary:	Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the 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 family members 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 a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. [provided by RefSeq, Jul 2008]
UniProt Code:	P17252
NCBI GenInfo Identifier:	317373571
NCBI Gene ID:	5578
NCBI Accession:	P17252.4
UniProt Secondary Accession:	P17252,Q15137, Q32M72, Q96RE4, B5BU22,
UniProt Related Accession:	P17252
Molecular Weight:	76,750 Da
NCBI Full Name:	Protein kinase C alpha type
NCBI Synonym Full Names:	protein kinase C, alpha
NCBI Official Symbol:	PRKCA
NCBI Official Synonym Symbols:	AAG6; PKCA; PRKACA; PKC-alpha
NCBI Protein Information:	protein kinase C alpha type; PKC-A; aging-associated gene 6
UniProt Protein Name:	Protein kinase C alpha type
Protein Family:	Protein kinase
UniProt Gene Name:	PRKCA
UniProt Entry Name:	KPCA_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.