Product Name:	Human PPP2R1A(Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform) ELISA Kit
Product Code:	HUFI03342
Size:	96 Assays
Alias:	PPP2R1A, PR65A
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PPP2R1A concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human PPP2R1A and the recovery rates were calculated by comparing the measured value to the expected amount of Human PPP2R1A in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PPP2R1A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
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

P30153

UniProt Protein Function:	PPP2R1A: The PR65 subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit. Required for proper chromosome segregation and for centromeric localization of SGOL1 in mitosis. Found in a complex with at least ARL2, PPP2CB, PPP2R1A, PPP2R2A, PPP2R5E and TBCD. PP2A consists of a common heterodimeric core enzyme, composed of PPP2CA a 36 kDa catalytic subunit (subunit C) and PPP2R1A a 65 kDa constant regulatory subunit (PR65 or subunit A), that associates with a variety of regulatory subunits. Proteins that associate with the core dimer include three families of regulatory subunits B (the R2/B/PR55/B55, R3/B''/PR72/PR130/PR59 and R5/B'/B56 families), the 48 kDa variable regulatory subunit, viral proteins, and cell signaling molecules. Interacts with IPO9. Interacts with TP53 and SGOL1. Interacts with PLA2G16; this interaction might decrease PP2A activity. Belongs to the phosphatase 2A regulatory subunit A family.
UniProt Protein Details:	Protein type:Protein phosphatase, regulatory subunit; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 19q13.41 Cellular Component: microtubule cytoskeleton; protein phosphatase type 2A complex; mitochondrion; membrane; nucleus; cytosol; chromosome, pericentric region Molecular Function:protein binding; protein heterodimerization activity; protein phosphatase type 2A regulator activity; protein serine/threonine phosphatase activity; antigen binding Biological Process: regulation of cell adhesion; second-messenger-mediated signaling; mitotic nuclear envelope reassembly; fibroblast growth factor receptor signaling pathway; apoptosis; organelle organization and biogenesis; RNA splicing; regulation of Wnt receptor signaling pathway; protein amino acid dephosphorylation; chromosome segregation; response to organic substance; regulation of transcription, DNA-dependent; regulation of growth; regulation of cell differentiation; mRNA catabolic process, nonsense-mediated decay; negative regulation of tyrosine phosphorylation of Stat3 protein; protein complex assembly; gene expression; mitotic cell cycle; negative regulation of cell growth; G2/M transition of mitotic cell cycle; ceramide metabolic process; inactivation of MAPK activity; regulation of DNA replication Disease: Mental Retardation, Autosomal Dominant 36
NCBI Summary:	This gene encodes a constant regulatory subunit of protein phosphatase 2. Protein phosphatase 2 is one of the four major Ser/Thr phosphatases, and it is implicated in the negative control of cell growth and division. It consists of a common heteromeric core enzyme, which is composed of a catalytic subunit and a constant regulatory subunit, that associates with a variety of regulatory subunits. The constant regulatory subunit A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit. This gene encodes an alpha isoform of the constant regulatory subunit A. Alternatively spliced transcript variants have been described. [provided by RefSeq, Apr 2010]
UniProt Code:	P30153
NCBI GenInfo Identifier:	143811355
NCBI Gene ID:	5518
NCBI Accession:	P30153.4
UniProt Secondary Accession:	P30153,Q13773, Q6ICQ3, Q96DH3,
UniProt Related Accession:	P30153
Molecular Weight:	589
NCBI Full Name:	Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform
NCBI Synonym Full Names:	protein phosphatase 2, regulatory subunit A, alpha
NCBI Official Symbol:	PPP2R1A
NCBI Official Synonym Symbols:	PR65A; PP2AAALPHA; PP2A-Aalpha
NCBI Protein Information:	serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform; PP2A subunit A isoform R1-alpha; PP2A subunit A isoform PR65-alpha; medium tumor antigen-associated 61 KDA protein; protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), alpha isoform; serine/threonine protein phosphatase 2A, 65 kDa regulatory subunit A, alpha isoform
UniProt Protein Name:	Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform
UniProt Synonym Protein Names:	Medium tumor antigen-associated 61 kDa protein; PP2A subunit A isoform PR65-alpha; PP2A subunit A isoform R1-alpha
UniProt Gene Name:	PPP2R1A
UniProt Entry Name:	2AAA_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 PPP2R1A(Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform) ELISA Kit

Description