Product Name:	Human BMP4 ELISA Kit
Product Code:	HUFI00042
Size:	96 Assays
Alias:	BMP-4, BMP2B, BMP2B1, MCOPS6, ZYME, DVR4, OFC11, ZYME
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human BMP-4 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 BMP-4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human BMP-4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-100	96
EDTA plasma(n=5)	86-102	91
UFH plasma(n=5)	85-100	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human BMP-4 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)	86-97%	86-103%	90-103%
EDTA plasma(n=5)	83-99%	87-101%	90-101%
UFH plasma(n=5)	86-98%	86-100%	82-100%

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

P12644

UniProt Protein Function:	BMP4: Induces cartilage and bone formation. Also act in mesoderm induction, tooth development, limb formation and fracture repair. Acts in concert with PTHLH/PTHRP to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction. Homodimer; disulfide-linked. Interacts with GREM2. Part of a complex consisting of TWSG1 and CHRD. Interacts with the serine proteases, HTRA1 and HTRA3; the interaction with either inhibits BMP4-mediated signaling. The HTRA protease activity is required for this inhibition. Interacts with SOSTDC1. Expressed in the lung and lower levels seen in the kidney. Present also in normal and neoplastic prostate tissues, and prostate cancer cell lines. Belongs to the TGF-beta family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 14q22.2 Cellular Component: extracellular region; extracellular space Molecular Function:chemoattractant activity; cytokine activity; protein binding; transforming growth factor beta receptor binding Biological Process: activation of MAPKK activity; alveolus development; blood vessel endothelial cell proliferation during sprouting angiogenesis; BMP signaling pathway; branching morphogenesis of a tube; chondrocyte differentiation; common-partner SMAD protein phosphorylation; endochondral ossification; hemopoietic progenitor cell differentiation; intermediate mesodermal cell differentiation; kidney development; lymphoid progenitor cell differentiation; macrophage differentiation; mesonephros development; monocyte differentiation; negative regulation of apoptosis; negative regulation of cell cycle; negative regulation of cell proliferation; negative regulation of immature T cell proliferation in the thymus; negative regulation of MAP kinase activity; negative regulation of mitosis; negative regulation of myoblast differentiation; negative regulation of phosphorylation; negative regulation of striated muscle development; negative regulation of T cell differentiation in the thymus; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; odontogenesis; osteoblast differentiation; positive regulation of apoptosis; positive regulation of BMP signaling pathway; positive regulation of bone mineralization; positive regulation of cardiac muscle fiber development; positive regulation of collagen biosynthetic process; positive regulation of endothelial cell proliferation; positive regulation of epidermal cell differentiation; positive regulation of epithelial cell proliferation; positive regulation of ossification; positive regulation of osteoblast differentiation; positive regulation of protein amino acid phosphorylation; positive regulation of protein binding; positive regulation of smooth muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; post-embryonic development; regulation of cell fate commitment; regulation of protein import into nucleus; smooth muscle development; smoothened signaling pathway; steroid hormone mediated signaling; telencephalon development; ureteric bud branching; ureteric bud development Disease: Microphthalmia, Syndromic 6; Orofacial Cleft 11
NCBI Summary:	This gene encodes a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Ligands of this family bind various TGF-beta receptors leading to recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein is proteolytically processed to generate each subunit of the disulfide-linked homodimer. This protein regulates heart development and adipogenesis. Mutations in this gene are associated with orofacial cleft and microphthalmia in human patients. The encoded protein may also be involved in the pathology of multiple cardiovascular diseases and human cancers. [provided by RefSeq, Jul 2016]
UniProt Code:	P12644
NCBI GenInfo Identifier:	115073
NCBI Gene ID:	652
NCBI Accession:	P12644.1
UniProt Secondary Accession:	P12644,Q9UM80,
UniProt Related Accession:	P12644
Molecular Weight:	47kDa
NCBI Full Name:	Bone morphogenetic protein 4
NCBI Synonym Full Names:	bone morphogenetic protein 4
NCBI Official Symbol:	BMP4
NCBI Official Synonym Symbols:	ZYME; BMP2B; OFC11; BMP2B1; MCOPS6
NCBI Protein Information:	bone morphogenetic protein 4
UniProt Protein Name:	Bone morphogenetic protein 4
UniProt Synonym Protein Names:	Bone morphogenetic protein 2B; BMP-2B
Protein Family:	Bone morphogenetic protein
UniProt Gene Name:	BMP4

*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.