Rat PPP3R1/Calcineurin B ELISA Kit- High Sensitivity

Product Name:	Rat PPP3R1/Calcineurin B ELISA Kit
Product Code:	RTFI00361
Size:	96 Assays
Target:	Rat Ppp3r1
Alias:	Ppp3r1, CALNB1, Cnb1, MCIP1, PP2B beta 1, PPP3R1, CNA2, CNB1, Protein phosphatase 2B regulatory subunit 1, protein phosphatase 2B regulatory subunit B alpha, protein phosphatase 3
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
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 Rat Ppp3r1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Ppp3r1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	97-102	99
EDTA plasma(n=5)	86-104	97
UFH plasma(n=5)	86-104	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Ppp3r1 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)	90-104%	85-103%	87-104%
EDTA plasma(n=5)	82-101%	91-101%	82-97%
UFH plasma(n=5)	82-100%	80-93%	81-91%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P63100

UniProt Protein Function:	p63: Acts as a sequence specific DNA binding transcriptional activator or repressor. The isoforms contain a varying set of transactivation and auto-regulating transactivation inhibiting domains thus showing an isoform specific activity. Isoform 2 activates RIPK4 transcription. May be required in conjunction with TP73/p73 for initiation of p53/TP53 dependent apoptosis in response to genotoxic insults and the presence of activated oncogenes. Involved in Notch signaling by probably inducing JAG1 and JAG2. Plays a role in the regulation of epithelial morphogenesis. The ratio of DeltaN-type and TA*-type isoforms may govern the maintenance of epithelial stem cell compartments and regulate the initiation of epithelial stratification from the undifferentiated embryonal ectoderm. Required for limb formation from the apical ectodermal ridge. Activates transcription of the p21 promoter. Binds DNA as a homotetramer. Isoform composition of the tetramer may determine transactivation activity. Isoforms Alpha and Gamma interact with HIPK2. Interacts with SSRP1, leading to stimulate coactivator activity. Isoform 1 and isoform 2 interact with WWP1. Interacts with PDS5A. Isoform 5 (via activation domain) interacts with NOC2L. Widely expressed, notably in heart, kidney, placenta, prostate, skeletal muscle, testis and thymus, although the precise isoform varies according to tissue type. Progenitor cell layers of skin, breast, eye and prostate express high levels of DeltaN-type isoforms. Isoform 10 is predominantly expressed in skin squamous cell carcinomas, but not in normal skin tissues. Belongs to the p53 family. 12 isoforms of the human protein are produced by alternative promoter.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 3q28 Cellular Component: nucleoplasm; transcription factor complex; rough endoplasmic reticulum; cytoplasm; dendrite; nuclear chromatin; nucleus; cytosol; chromatin Molecular Function:identical protein binding; protein binding; DNA binding; p53 binding; sequence-specific DNA binding; metal ion binding; double-stranded DNA binding; damaged DNA binding; WW domain binding; chromatin binding; transcription factor activity Biological Process: G1 DNA damage checkpoint; ectoderm and mesoderm interaction; apoptosis; positive regulation of transcription, DNA-dependent; cloacal septation; epidermal cell division; negative regulation of transcription from RNA polymerase II promoter; regulation of caspase activity; protein homotetramerization; polarized epithelial cell differentiation; smooth muscle development; sympathetic nervous system development; DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator; regulation of neuron apoptosis; positive regulation of mesenchymal cell proliferation; epithelial cell development; response to gamma radiation; establishment of planar polarity; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; skeletal development; female genitalia morphogenesis; positive regulation of Notch signaling pathway; proximal/distal pattern formation; response to X-ray; embryonic limb morphogenesis; regulation of epidermal cell division; Notch signaling pathway; hair follicle morphogenesis; transcription, DNA-dependent; urinary bladder development; negative regulation of keratinocyte differentiation; multicellular organismal aging; keratinocyte proliferation; replicative cell aging; odontogenesis of dentine-containing teeth; keratinocyte differentiation; chromatin remodeling; positive regulation of osteoblast differentiation; neuron apoptosis; spermatogenesis; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; response to DNA damage stimulus; negative regulation of apoptosis Disease: Ankyloblepharon-ectodermal Defects-cleft Lip/palate; Rapp-hodgkin Syndrome; Adult Syndrome; Ectrodactyly, Ectodermal Dysplasia, And Cleft Lip/palate Syndrome 3; Split-hand/foot Malformation 4; Limb-mammary Syndrome
NCBI Summary:	This gene encodes a member of the p53 family of transcription factors. An animal model, p63 -/- mice, has been useful in defining the role this protein plays in the development and maintenance of stratified epithelial tissues. p63 -/- mice have several developmental defects which include the lack of limbs and other tissues, such as teeth and mammary glands, which develop as a result of interactions between mesenchyme and epithelium. Mutations in this gene are associated with ectodermal dysplasia, and cleft lip/palate syndrome 3 (EEC3); split-hand/foot malformation 4 (SHFM4); ankyloblepharon-ectodermal defects-cleft lip/palate; ADULT syndrome (acro-dermato-ungual-lacrimal-tooth); limb-mammary syndrome; Rap-Hodgkin syndrome (RHS); and orofacial cleft 8. Both alternative splicing and the use of alternative promoters results in multiple transcript variants encoding different proteins. Many transcripts encoding different proteins have been reported but the biological validity and the full-length nature of these variants have not been determined. [provided by RefSeq, Jul 2008]
UniProt Code:	P63100
NCBI GenInfo Identifier:	31543818
NCBI Gene ID:	8626
NCBI Accession:	NP_003713
UniProt Secondary Accession:	P63100,O75080, O75195, O75922, O76078, Q6VEG2, Q6VEG3 Q6VEG4, Q6VFJ1, Q6VFJ2, Q6VFJ3, Q6VH20,
UniProt Related Accession:	Q9H3D4
Molecular Weight:	77kd (Predicted)
NCBI Full Name:	tumor protein 63 isoform 1
NCBI Synonym Full Names:	tumor protein p63
NCBI Official Symbol:	TP63
NCBI Official Synonym Symbols:	AIS; KET; LMS; NBP; RHS; p40; p51; p63; EEC3; OFC8; p73H; p73L; SHFM4; TP53L; TP73L; p53CP; TP53CP; B(p51A); B(p51B)
NCBI Protein Information:	tumor protein 63
UniProt Protein Name:	Tumor protein 63
UniProt Synonym Protein Names:	Chronic ulcerative stomatitis protein; CUSP; Keratinocyte transcription factor KET; Transformation-related protein 63; TP63; Tumor protein p73-like; p73L; p40; p51
UniProt Gene Name:	TP63
UniProt Entry Name:	P63_HUMAN

Step	Procedure
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.