Human PRKACA ELISA Kit (HUEB2037)- High Sensitivity

Product Name:	Human cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) ELISA Kit
SKU:	HUEB2037
Size:	96T
Target:	Human cAMP-dependent protein kinase catalytic subunit alpha (PRKACA)
Synonyms:	PKA C-alpha, PKACA
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.78-50ng/mL
Sensitivity:	0.34ng/mL

Intra CV:

4.1%

Inter CV:

7.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	100-110%	106-116%	81-91%	83-93%
EDTA Plasma(N=5)	97-109%	100-112%	93-105%	99-108%
Heparin Plasma(N=5)	101-111%	94-104%	114-124%	80-92%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

Phosphorylates a large number of substrates in the cytoplasm and the nucleus. Regulates the abundance of compartmentalized pools of its regulatory subunits through phosphorylation of PJA2 which binds and ubiquitinates these subunits, leading to their subsequent proteolysis. Phosphorylates CDC25B, ABL1, NFKB1, CLDN3, PSMC5/RPT6, PJA2, RYR2, RORA and VASP. RORA is activated by phosphorylation. Required for glucose-mediated adipogenic differentiation increase and osteogenic differentiation inhibition from osteoblasts. Involved in the regulation of platelets in response to thrombin and collagen; maintains circulating platelets in a resting state by phosphorylating proteins in numerous platelet inhibitory pathways when in complex with NF-kappa-B (NFKB1 and NFKB2) and I-kappa-B-alpha (NFKBIA), but thrombin and collagen disrupt these complexes and free active PRKACA stimulates platelets and leads to platelet aggregation by phosphorylating VASP. Prevents the antiproliferative and anti-invasive effects of alpha-difluoromethylornithine in breast cancer cells when activated. RYR2 channel activity is potentiated by phosphorylation in presence of luminal Ca(2+), leading to reduced amplitude and increased frequency of store overload-induced Ca(2+) release (SOICR) characterized by an increased rate of Ca(2+) release and propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). PSMC5/RPT6 activation by phosphorylation stimulates proteasome. Negatively regulates tight junctions (TJs) in ovarian cancer cells via CLDN3 phosphorylation. NFKB1 phosphorylation promotes NF-kappa-B p50-p50 DNA binding. Involved in embryonic development by down-regulating the Hedgehog (Hh) signaling pathway that determines embryo pattern formation and morphogenesis. Prevents meiosis resumption in prophase-arrested oocytes via CDC25B inactivation by phosphorylation. May also regulate rapid eye movement (REM) sleep in the pedunculopontine tegmental (PPT). Phosphorylates APOBEC3G and AICDA. Isoform 2 phosphorylates and activates ABL1 in sperm flagellum to promote spermatozoa capacitation. Phosphorylates HSF1; this phosphorylation promotes HSF1 nuclear localization and transcriptional activity upon heat shock (PubMed:21085490).

Uniprot:	P17612
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human cAMP-dependent protein kinase catalytic subunit alpha
Sub Unit:	A number of inactive tetrameric holoenzymes are produced by the combination of homo- or heterodimers of the different regulatory subunits associated with two catalytic subunits. cAMP causes the dissociation of the inactive holoenzyme into a dimer of regulatory subunits bound to four cAMP and two free monomeric catalytic subunits. The cAMP-dependent protein kinase catalytic subunit binds PJA2. Both isoforms 1 and 2 forms activate cAMP-sensitive PKAI and PKAII holoenzymes by interacting with regulatory subunit (R) of PKA, PRKAR1A/PKR1 and PRKAR2A/PKR2, respectively. Interacts with NFKB1, NFKB2 and NFKBIA in platelets; these interactions are disrupted by thrombin and collagen. Binds to ABL1 in spermatozoa and with CDC25B in oocytes. Interacts with APOBEC3G and AICDA. Interacts with RAB13; downstream effector of RAB13 involved in tight junction assembly. Found in a complex at least composed of MROH2B, PRKACA isoform 2 and TCP11 (By similarity). Interacts with MROH2B (By similarity). Isoform 2 interacts with TCP11 (By similarity). Interacts with HSF1 (PubMed:21085490).
Research Area:	Cardiovascular
Subcellular Location:	Isoform 2 Cell projection Cilium Flagellum Cytoplasmic vesicle Secretory vesicle Acrosome Expressed in the midpiece region of the sperm flagellum (PubMed:10906071). Colocalizes with MROH2B and TCP11 on the acrosome and tail regions in round spermatids and spermatozoa regardless of the capacitation status of the sperm (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PKACA: catalytic subunit of cAMP-dependent protein kinase alpha, an AGC kinase. A number of inactive tetrameric holoenzymes are produced by the combination of homo- or heterodimers of the different regulatory subunits associated with two catalytic subunits. cAMP causes the dissociation of the inactive holoenzyme into a dimer of regulatory subunits bound to four cAMP and two free monomeric catalytic subunits. Two splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; EC 2.7.11.11; AGC group; PKA family Chromosomal Location of Human Ortholog: 19p13.1 Cellular Component: centrosome; membrane; mitochondrion; plasma membrane; cytosol; nucleus; neuromuscular junction; AMP-activated protein kinase complex; cAMP-dependent protein kinase complex Molecular Function:protein binding; cAMP-dependent protein kinase activity; ubiquitin protein ligase binding; protein serine/threonine/tyrosine kinase activity; protein kinase binding; ATP binding Biological Process: nerve growth factor receptor signaling pathway; protein amino acid autophosphorylation; water transport; pathogenesis; signal transduction; protein amino acid phosphorylation; positive regulation of protein export from nucleus; triacylglycerol catabolic process; G2/M transition of mitotic cell cycle; transmembrane transport; sperm capacitation; regulation of synaptic transmission; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; cytosolic calcium ion homeostasis; regulation of osteoblast differentiation; regulation of heart rate; organelle organization and biogenesis; activation of protein kinase A; peptidyl-threonine phosphorylation; glucose metabolic process; gluconeogenesis; peptidyl-serine phosphorylation; mesoderm formation; phospholipase C activation; regulation of protein binding; neural tube closure; carbohydrate metabolic process; energy reserve metabolic process; innate immune response; renal water homeostasis; mitotic cell cycle; blood coagulation; regulation of insulin secretion
NCBI Summary:	cAMP is a signaling molecule important for a variety of cellular functions. cAMP exerts its effects by activating the cAMP-dependent protein kinase, which transduces the signal through phosphorylation of different target proteins. The inactive kinase holoenzyme is a tetramer composed of two regulatory and two catalytic subunits. cAMP causes the dissociation of the inactive holoenzyme into a dimer of regulatory subunits bound to four cAMP and two free monomeric catalytic subunits. Four different regulatory subunits and three catalytic subunits have been identified in humans. The protein encoded by this gene is a member of the Ser/Thr protein kinase family and is a catalytic subunit of cAMP-dependent protein kinase. Alternatively spliced transcript variants encoding distinct isoforms have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:	P17612
NCBI GenInfo Identifier:	125205
NCBI Gene ID:	5566
NCBI Accession:	P17612.2
UniProt Secondary Accession:	P17612,Q32P54, Q9H2Y0, Q9NRB4, Q9NRH9,
UniProt Related Accession:	P17612
Molecular Weight:	39,953 Da
NCBI Full Name:	cAMP-dependent protein kinase catalytic subunit alpha
NCBI Synonym Full Names:	protein kinase, cAMP-dependent, catalytic, alpha
NCBI Official Symbol:	PRKACA
NCBI Official Synonym Symbols:	PKACA
NCBI Protein Information:	cAMP-dependent protein kinase catalytic subunit alpha; PKA C-alpha; protein kinase A catalytic subunit; cAMP-dependent protein kinase catalytic subunit alpha, isoform 1
UniProt Protein Name:	cAMP-dependent protein kinase catalytic subunit alpha
Protein Family:	cAMP-dependent protein kinase
UniProt Gene Name:	PRKACA
UniProt Entry Name:	KAPCA_HUMAN

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 PRKACA ELISA Kit