Human BMPR1A ELISA Kit (HUEB0405)- High Sensitivity

Product Name:	Human Bone morphogenetic protein receptor type-1A (BMPR1A) ELISA Kit
SKU:	HUEB0405
Size:	96T
Target:	Human Bone morphogenetic protein receptor type-1A (BMPR1A)
Synonyms:	Activin receptor-like kinase 3, Serine/threonine-protein kinase receptor R5, ALK-3, SKR5, CD292, BMP type-1A receptor, ACVRLK3, ALK3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.11ng/mL

Intra CV:

5.6%

Inter CV:

9.9%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	82-93%	103-112%	93-103%	81-93%
EDTA Plasma(N=5)	92-101%	94-104%	102-111%	87-97%
Heparin Plasma(N=5)	95-106%	90-101%	95-107%	104-116%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	107	101-113
Plasma	109	103-115

Function:

On ligand binding, forms a receptor complex consisting of two type II and two type I transmembrane serine/threonine kinases. Type II receptors phosphorylate and activate type I receptors which autophosphorylate, then bind and activate SMAD transcriptional regulators. Receptor for BMP2, BMP4, GDF5 and GDF6. Positively regulates chondrocyte differentiation through GDF5 interaction. Mediates induction of adipogenesis by GDF6.

Uniprot:	P36894
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Bone morphogenetic protein receptor type-1A
Sub Unit:	Interacts with BMP2 (PubMed:10881198, PubMed:18937504). Interacts with low affinity with GDF5; positively regulates chondrocyte differentiation (PubMed:24098149).
Research Area:	Stem Cells
Subcellular Location:	Membrane Single-pass type I membrane protein
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	BMPR1A: a serine/threonine-protein kinase receptor for Bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4). Defects in BMPR1A are a cause of juvenile polyposis syndrome (JPS) and Cowden disease (CD), a cancer syndrome characterized by multiple hamartomas and by a high risk for breast, thyroid and endometriel cancers.
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (receptor); Kinase, protein; Membrane protein, integral; Protein kinase, TKL; EC 2.7.11.30; TKL group; STKR family; Type1 subfamily Chromosomal Location of Human Ortholog: 10q22.3 Cellular Component: cell soma; dendrite; integral to membrane; plasma membrane; caveola Molecular Function:transforming growth factor beta receptor activity; protein serine/threonine kinase activity; protein binding; protein homodimerization activity; metal ion binding; SMAD binding; ATP binding; transmembrane receptor protein serine/threonine kinase activity; receptor signaling protein serine/threonine kinase activity Biological Process: neural plate mediolateral pattern formation; transcription from RNA polymerase II promoter; developmental growth; hindlimb morphogenesis; neural crest cell development; positive regulation of transcription, DNA-dependent; paraxial mesoderm structural organization; mesendoderm development; dorsal/ventral axis specification; palate development; protein amino acid phosphorylation; negative regulation of neurogenesis; BMP signaling pathway; transforming growth factor beta receptor signaling pathway; positive regulation of mesenchymal cell proliferation; ectoderm development; Mullerian duct regression; somitogenesis; in utero embryonic development; lateral mesoderm development; stem cell maintenance; positive regulation of bone mineralization; odontogenesis of dentine-containing teeth; positive regulation of osteoblast differentiation; mesoderm formation; pituitary gland development; cartilage development; embryonic organ development; immune response; embryonic digit morphogenesis; regulation of lateral mesodermal cell fate specification; positive regulation of epithelial cell proliferation; lung development Disease: Juvenile Polyposis Syndrome; Polyposis Syndrome, Hereditary Mixed, 2
NCBI Summary:	The bone morphogenetic protein (BMP) receptors are a family of transmembrane serine/threonine kinases that include the type I receptors BMPR1A and BMPR1B and the type II receptor BMPR2. These receptors are also closely related to the activin receptors, ACVR1 and ACVR2. The ligands of these receptors are members of the TGF-beta superfamily. TGF-betas and activins transduce their signals through the formation of heteromeric complexes with 2 different types of serine (threonine) kinase receptors: type I receptors of about 50-55 kD and type II receptors of about 70-80 kD. Type II receptors bind ligands in the absence of type I receptors, but they require their respective type I receptors for signaling, whereas type I receptors require their respective type II receptors for ligand binding. [provided by RefSeq, Jul 2008]
UniProt Code:	P36894
NCBI GenInfo Identifier:	61252444
NCBI Gene ID:	657
NCBI Accession:	P36894.2
UniProt Secondary Accession:	P36894,Q8NEN8, A8K6U9,
UniProt Related Accession:	P36894
Molecular Weight:	Calculated: 60kDaObserved: 69kDa
NCBI Full Name:	Bone morphogenetic protein receptor type-1A
NCBI Synonym Full Names:	bone morphogenetic protein receptor, type IA
NCBI Official Symbol:	BMPR1A
NCBI Official Synonym Symbols:	ALK3; SKR5; CD292; ACVRLK3; 10q23del
NCBI Protein Information:	bone morphogenetic protein receptor type-1A; ALK-3; BMPR-1A; BMP type-1A receptor; activin receptor-like kinase 3; activin A receptor, type II-like kinase 3; serine/threonine-protein kinase receptor R5
UniProt Protein Name:	Bone morphogenetic protein receptor type-1A
UniProt Synonym Protein Names:	Activin receptor-like kinase 3; ALK-3; Serine/threonine-protein kinase receptor R5; SKR5; CD_antigen: CD292
Protein Family:	Bone morphogenetic protein receptor
UniProt Gene Name:	BMPR1A
UniProt Entry Name:	BMR1A_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	Antibodies
Human BMPR1A / ALK3 ELISA Kit	Anti-BMPR1A Antibody (CAB1816)
Human BMPR1A (Bone Morphogenetic Protein Receptor, type IA) ELISA Kit (HUES01762)	BMPR1A Antibody (PACO02196)
	BMP7 Antibody (PACO08029)
	BMP3 Antibody, Biotin conjugated (PACO25271)
	BMPR1A Antibody (PACO25272)