Product Name:	Human TGF-beta ELISA Kit
Product Code:	HUFI00248
Size:	96 Assays
Alias:	TGF-beta1, Transforming Growth Factor Beta 1, TGF-B1, TGFB, TGFbeta, CED, DPD1, TGFbeta1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TGF-beta1 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 TGF-beta1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TGF-beta1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-102	95
EDTA plasma(n=5)	96-104	99
UFH plasma(n=5)	92-102	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TGF-beta1 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)	89-95%	89-103%	85-103%
EDTA plasma(n=5)	92-101%	86-101%	86-95%
UFH plasma(n=5)	81-97%	80-99%	85-100%

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

P01137

UniProt Protein Function:	TGFB1: Multifunctional protein that controls proliferation, differentiation and other functions in many cell types. Many cells synthesize TGFB1 and have specific receptors for it. It positively and negatively regulates many other growth factors. It plays an important role in bone remodeling as it is a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts. Homodimer; disulfide-linked, or heterodimer with TGFB2. Secreted and stored as a biologically inactive form in the extracellular matrix in a 290 kDa complex (large latent TGF-beta1 complex) containing the TGFB1 homodimer, the latency-associated peptide (LAP), and the latent TGFB1 binding protein-1 (LTBP1). The complex without LTBP1 is known as the'small latent TGF-beta1 complex'. Dissociation of the TGFB1 from LAP is required for growth factor activation and biological activity. Release of the large latent TGF-beta1 complex from the extracellular matrix is carried out by the matrix metalloproteinase MMP3. May interact with THSD4; this interaction may lead to sequestration by FBN1 microfibril assembly and attenuation of TGFB signaling. Interacts with the serine proteases, HTRA1 and HTRA3: the interaction with either inhibits TGFB1-mediated signaling. The HTRA protease activity is required for this inhibition. Interacts with CD109, DPT and ASPN. Activated in vitro at pH below 3.5 and over 12.5. Highly expressed in bone. Abundantly expressed in articular cartilage and chondrocytes and is increased in osteoarthritis (OA). Co-localizes with ASPN in chondrocytes within OA lesions of articular cartilage. Belongs to the TGF-beta family.
UniProt Protein Details:	Protein type:Secreted; Motility/polarity/chemotaxis; Secreted, signal peptide Chromosomal Location of Human Ortholog: 19q13.1 Cellular Component: extracellular space; proteinaceous extracellular matrix; microvillus; cell surface; cell soma; axon; Golgi lumen; cytoplasm; plasma membrane; extracellular region; nucleus Molecular Function:protein binding; enzyme binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; punt binding; cytokine activity; protein N-terminus binding; glycoprotein binding; antigen binding Biological Process: extracellular matrix organization and biogenesis; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; SMAD protein nuclear translocation; female pregnancy; positive regulation of protein amino acid dephosphorylation; activation of NF-kappaB transcription factor; regulation of protein import into nucleus; positive regulation of MAP kinase activity; connective tissue replacement during inflammatory response; regulation of transforming growth factor beta receptor signaling pathway; negative regulation of ossification; cell cycle arrest; positive regulation of isotype switching to IgA isotypes; inner ear development; regulatory T cell differentiation; positive regulation of interleukin-17 production; response to drug; positive regulation of smooth muscle cell differentiation; positive regulation of chemotaxis; active induction of host immune response by virus; positive regulation of blood vessel endothelial cell migration; regulation of sodium ion transport; negative regulation of blood vessel endothelial cell migration; negative regulation of fat cell differentiation; lymph node development; positive regulation of protein secretion; positive regulation of transcription from RNA polymerase II promoter; response to progesterone stimulus; endoderm development; positive regulation of odontogenesis; myelination; negative regulation of phagocytosis; evasion of host defenses by virus; positive regulation of cellular protein metabolic process; myeloid dendritic cell differentiation; negative regulation of transcription from RNA polymerase II promoter; phosphate metabolic process; negative regulation of cell proliferation; negative regulation of T cell proliferation; ureteric bud development; regulation of DNA binding; negative regulation of release of sequestered calcium ion into cytosol; positive regulation of cell proliferation; salivary gland morphogenesis; protein kinase B signaling cascade; protein export from nucleus; inflammatory response; aging; positive regulation of exit from mitosis; epidermal growth factor receptor signaling pathway; mitotic cell cycle checkpoint; common-partner SMAD protein phosphorylation; positive regulation of phosphoinositide 3-kinase activity; positive regulation of bone mineralization; positive regulation of peptidyl-serine phosphorylation; SMAD protein complex assembly; positive regulation of protein kinase B signaling cascade; positive regulation of protein complex assembly; positive regulation of protein import into nucleus; response to hypoxia; epithelial to mesenchymal transition; negative regulation of cell growth; negative regulation of cell-cell adhesion; negative regulation of transforming growth factor beta receptor signaling pathway; negative regulation of skeletal muscle development; mononuclear cell proliferation; protein amino acid phosphorylation; regulation of cell migration; hyaluronan catabolic process; regulation of apoptosis; response to vitamin D; negative regulation of neuroblast proliferation; positive regulation of superoxide release; receptor catabolic process; transforming growth factor beta receptor signaling pathway; germ cell migration; response to glucose stimulus; chondrocyte differentiation; T cell homeostasis; defense response to fungus, incompatible interaction; negative regulation of mitotic cell cycle; cell growth; tolerance induction to self antigen; regulation of striated muscle development; platelet activation; organ regeneration; negative regulation of DNA replication; virus-host interaction; hemopoietic progenitor cell differentiation; negative regulation of transcription, DNA-dependent; positive regulation of epithelial cell proliferation; positive regulation of collagen biosynthetic process; viral infectious cycle; response to estradiol stimulus; negative regulation of cell cycle; positive regulation of histone deacetylation; response to radiation; platelet degranulation; negative regulation of protein amino acid phosphorylation; response to wounding; lipopolysaccharide-mediated signaling pathway; adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains; negative regulation of epithelial cell proliferation; intercellular junction assembly and maintenance; regulation of binding; MAPKKK cascade; cellular calcium ion homeostasis; gut development; protein import into nucleus, translocation; ATP biosynthetic process; positive regulation of histone acetylation; positive regulation of protein amino acid phosphorylation; negative regulation of myoblast differentiation; blood coagulation; positive regulation of cell migration Disease: Camurati-engelmann Disease; Cystic Fibrosis
NCBI Summary:	This gene encodes a member of the transforming growth factor beta (TGFB) family of cytokines, which are multifunctional peptides that regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. Many cells have TGFB receptors, and the protein positively and negatively regulates many other growth factors. The secreted protein is cleaved into a latency-associated peptide (LAP) and a mature TGFB1 peptide, and is found in either a latent form composed of a TGFB1 homodimer, a LAP homodimer, and a latent TGFB1-binding protein, or in an active form composed of a TGFB1 homodimer. The mature peptide may also form heterodimers with other TGFB family members. This gene is frequently upregulated in tumor cells, and mutations in this gene result in Camurati-Engelmann disease.[provided by RefSeq, Oct 2009]
UniProt Code:	P01137
NCBI GenInfo Identifier:	135674
NCBI Gene ID:	7040
NCBI Accession:	P01137.2
UniProt Secondary Accession:	P01137,Q9UCG4, A8K792,
UniProt Related Accession:	P01137
Molecular Weight:	44,341 Da
NCBI Full Name:	Transforming growth factor beta-1
NCBI Synonym Full Names:	transforming growth factor, beta 1
NCBI Official Symbol:	TGFB1
NCBI Official Synonym Symbols:	CED; LAP; DPD1; TGFB; TGFbeta
NCBI Protein Information:	transforming growth factor beta-1; TGF-beta-1; latency-associated peptide; prepro-transforming growth factor beta-1
UniProt Protein Name:	Transforming growth factor beta-1
UniProt Synonym Protein Names:
Protein Family:	Transforming growth factor
UniProt Gene Name:	TGFB1
UniProt Entry Name:	TGFB1_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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