Human PPP1CA ELISA Kit (HUEB2672)- High Sensitivity

Product Name:	Human Serine/threonine-protein phosphatase PP1-alpha catalytic subunit (PPP1CA) ELISA Kit
SKU:	HUEB2672
Size:	96T
Target:	Human Serine/threonine-protein phosphatase PP1-alpha catalytic subunit (PPP1CA)
Synonyms:	PP-1A, PPP1A
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.15ng/mL

Intra CV:

4.6%

Inter CV:

8.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	94-104%	99-109%	93-93%	104-114%
EDTA Plasma(N=5)	103-103%	84-94%	80-90%	106-115%
Heparin Plasma(N=5)	96-107%	95-105%	101-111%	101-111%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	93	87-99
Plasma	95	89-101

Function:

Protein phosphatase that associates with over 200 regulatory proteins to form highly specific holoenzymes which dephosphorylate hundreds of biological targets. Protein phosphatase 1 (PP1) is essential for cell division, and participates in the regulation of glycogen metabolism, muscle contractility and protein synthesis. Involved in regulation of ionic conductances and long-term synaptic plasticity. May play an important role in dephosphorylating substrates such as the postsynaptic density-associated Ca(2+)/calmodulin dependent protein kinase II. Component of the PTW/PP1 phosphatase complex, which plays a role in the control of chromatin structure and cell cycle progression during the transition from mitosis into interphase. Regulates NEK2 function in terms of kinase activity and centrosome number and splitting, both in the presence and absence of radiation-induced DNA damage. Regulator of neural tube and optic fissure closure, and enteric neural crest cell (ENCCs) migration during development. In balance with CSNK1D and CSNK1E, determines the circadian period length, through the regulation of the speed and rhythmicity of PER1 and PER2 phosphorylation. May dephosphorylate CSNK1D and CSNK1E. Dephosphorylates the 'Ser-418' residue of FOXP3 in regulatory T-cells (Treg) from patients with rheumatoid arthritis, thereby inactivating FOXP3 and rendering Treg cells functionally defective (PubMed:23396208). Dephosphorylates CENPA (PubMed:25556658). Dephosphorylates the 'Ser-139' residue of ATG16L1 causing dissociation of ATG12-ATG5-ATG16L1 complex, thereby inhibiting autophagy (PubMed:26083323).

Uniprot:	P62136
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
Sub Unit:	PP1 comprises a catalytic subunit, PPP1CA, PPP1CB or PPP1CC, which is folded into its native form by inhibitor 2 and glycogen synthetase kinase 3, and then complexed to one or several targeting or regulatory subunits. PPP1R12A, PPP1R12B and PPP1R12C mediate binding to myosin. PPP1R3A (in skeletal muscle), PPP1R3B (in liver), PPP1R3C, PPP1R3D and PPP1R3F (in brain) mediate binding to glycogen. Interacts with PPP1R39 (By similarity). Interacts with BTBD10 (By similarity). Interacts with KCTD20 (By similarity). Interacts with PPP1R9A and PPP1R9B. Part of a complex containing PPP1R15B, PP1 and NCK1/2. Interacts with PHACTR4; which acts as an activator of PP1 activity (By similarity). Interacts with PPP1R15A and PPP1R15B; the interactions mediate binding to EIF2S1. Component of the MLL5-L complex, at least composed of KMT2E/MLL5, STK38, PPP1CA, PPP1CB, PPP1CC, HCFC1, ACTB and OGT. Interacts with PPP1R7. Interacts with YLPM1. Forms a complex with ILF2, ILF3, YLPM1, KHDRBS1, RBMX and NCOA5. Interacts with NOM1 and PPP1R8. Interacts with HHV-1 ICP34.5. Interacts with PPP1R16B. Interacts with RPSA only in the presence of PPP1R16B. Component of the PTW/PP1 phosphatase complex, composed of PPP1R10/PNUTS, TOX4, WDR82, and PPP1CA or PPP1CB or PPP1CC. Interacts with PPP1R10/PNUTS and PPP1R8. Interacts with WDR82 in the presence of PPP1R10/PNUTS. Interacts with TRIM28; the interaction dephosphorylates TRIM28 on 'Ser-824' and forms a complex at the p21 promoter site. Interacts with isoform 1 and isoform 4 of NEK2. Interacts with FER; this promotes phosphorylation at Thr-320. Interacts with DAB2; the interaction is mutually exclusive with the AXIN1:PPP1CA interaction. Interacts with FOXP3 (PubMed:23396208). Interacts with CENPA (PubMed:25556658). Interacts with ATG16L1 (PubMed:26083323).
Research Area:	Cardiovascular
Subcellular Location:	Cytoplasm Nucleus Nucleus Nucleoplasm Nucleus Nucleolus Primarily nuclear and largely excluded from the nucleolus. Highly mobile in cells and can be relocalized through interaction with targeting subunits. NOM1 plays a role in targeting this protein to the nucleolus. In the presence of PPP1R8 relocalizes from the nucleus to nuclear speckles.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PPP1CA: is a catalytic subunit of protein phosphatase 1 (PP1), aubiquitous cytosolic serine-threonine phosphatase. Composed of a catalytic subunit (PPP1CA, PPP1CB or PPP1CC) which is folded into its native form by inhibitor 2 and glycogen synthetase kinase 3, and then complexed to one or several targeting (or regulatory) subunits: PPP1R12A and PPP1R12B mediate binding to myosin; PPP1R3A, PPP1R3B, PPP1R3C and PPP1R3D mediate binding to glycogen; and PPP1R15A and PPP1R15B mediate binding to EIF2S1. PP1 is essential for cell division, plays critical roles in many cellular processes including glycogen metabolism, muscle contractility and protein synthesis, and is involved in the regulation of ionic conductances and long-term synaptic plasticity. Inhibits the synthesis of proteins involved in synaptic plasticity. Binds to the transcription factor cyclic AMP-dependent response element binding (CREB) and dephosphorylates it. May regulate chromatin condensation through regulation of histone H3 phosphorylation. Differentially expressed in gastric cancer. May participate in the neurodegenerative progress in Alzheimer's disease. Down-regulated in lung squamous cell carcinoma.
UniProt Protein Details:	Protein type:EC 3.1.3.16; Nucleolus; Protein phosphatase, Ser/Thr (non-receptor) Chromosomal Location of Human Ortholog: 11q13.2 Cellular Component: cell-cell adherens junction; cytoplasm; cytosol; nuclear chromosome, telomeric region; nucleoplasm; nucleus; plasma membrane Molecular Function:phosphoprotein phosphatase activity; phosphoric monoester hydrolase activity; protein binding Biological Process: circadian regulation of gene expression; dephosphorylation; entrainment of circadian clock by photoperiod; negative regulation of protein binding; protein amino acid dephosphorylation; regulation of circadian rhythm
NCBI Summary:	The protein encoded by this gene is one of the three catalytic subunits of protein phosphatase 1 (PP1). PP1 is a serine/threonine specific protein phosphatase known to be involved in the regulation of a variety of cellular processes, such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and HIV-1 viral transcription. Increased PP1 activity has been observed in the end stage of heart failure. Studies in both human and mice suggest that PP1 is an important regulator of cardiac function. Mouse studies also suggest that PP1 functions as a suppressor of learning and memory. Three alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P62136
NCBI GenInfo Identifier:	49065811
NCBI Gene ID:	5499
NCBI Accession:	P62136.1
UniProt Secondary Accession:	P62136,P08129, P20653, P22802, Q07161, A6NNR3, B2R908
UniProt Related Accession:	P62136
Molecular Weight:	Calculated MW: 32kDa/37kDa/38kDaObserved MW: 38kDa
NCBI Full Name:	Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
NCBI Synonym Full Names:	protein phosphatase 1 catalytic subunit alpha
NCBI Official Symbol:	PPP1CA
NCBI Official Synonym Symbols:	PP1A; PP-1A; PPP1A; PP1alpha
NCBI Protein Information:	serine/threonine-protein phosphatase PP1-alpha catalytic subunit
UniProt Protein Name:	Serine/threonine-protein phosphatase PP1-alpha catalytic subunit
Protein Family:	Serine/threonine-protein phosphatase
UniProt Gene Name:	PPP1CA

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA

Human PPP1CA / Serine / threonine-protein phosphatase PP1-alpha catalytic subunit ELISA Kit