Human TGFB2/TGF-beta 2 ELISA Kit (HUFI00249)

Product Name:	Human TGFB2/TGF-beta 2 ELISA Kit
Product Code:	HUFI00249
Size:	96 Assays
Alias:	TGF-beta2, Transforming Growth Factor beta2, TGF-beta2, TGFbeta2, TGFB2, BSC-1 cell growth inhibitor, Cetermin, G-TSF, Polyergin, polyergin, TGF-beta2, TGF-beta-2, transforming growth factor beta-2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TGF-beta2 concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human TGF-beta2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TGF-beta2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-105	98
EDTA plasma(n=5)	87-104	97
UFH plasma(n=5)	86-103	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TGF-beta2 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)	87-104%	97-104%	93-104%
EDTA plasma(n=5)	83-98%	87-100%	82-100%
UFH plasma(n=5)	80-99%	83-100%	80-98%

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

P61812

UniProt Protein Function:	TGFB2: TGF-beta 2 has suppressive effects on interleukin-2 dependent T-cell growth. Homodimer; disulfide-linked. Heterodimers with TGFB1 and with TGFB3 have been found in bone. Interacts with the serine proteases, HTRA1 and HTRA3. Interacts with ASPN. Belongs to the TGF-beta family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell development/differentiation; Motility/polarity/chemotaxis; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 1q41 Cellular Component: extracellular matrix; extracellular space; cell soma; axon; extracellular region; endosome Molecular Function:protein binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; beta-amyloid binding; punt binding; cytokine activity; transforming growth factor beta receptor binding; receptor signaling protein serine/threonine kinase activity; receptor binding Biological Process: heart morphogenesis; extracellular matrix organization and biogenesis; collagen fibril organization; catagen; heart development; dopamine biosynthetic process; SMAD protein nuclear translocation; protein amino acid phosphorylation; cell-cell signaling; hair follicle development; transforming growth factor beta receptor signaling pathway; somatic stem cell division; cell cycle arrest; cell growth; embryonic gut development; cartilage condensation; response to drug; platelet activation; neutrophil chemotaxis; negative regulation of immune response; neuron fate commitment; positive regulation of cell cycle; positive regulation of catagen; positive regulation of cell growth; positive regulation of phosphoinositide 3-kinase cascade; cardioblast differentiation; positive regulation of protein secretion; positive regulation of cell division; activation of protein kinase activity; neuron development; response to progesterone stimulus; positive regulation of heart contraction; cell death; axon guidance; positive regulation of immune response; wound healing; cell morphogenesis; cardiac muscle cell proliferation; positive regulation of stress-activated MAPK cascade; odontogenesis; negative regulation of cell proliferation; platelet degranulation; positive regulation of neuron apoptosis; salivary gland morphogenesis; positive regulation of cell proliferation; response to wounding; hemopoiesis; angiogenesis; positive regulation of integrin biosynthetic process; negative regulation of epithelial cell proliferation; uterine wall breakdown; intercellular junction assembly and maintenance; cell migration; regulation of transforming growth factor-beta2 production; hair follicle morphogenesis; positive regulation of cell adhesion mediated by integrin; glial cell migration; positive regulation of ossification; cell proliferation; embryonic development; eye development; generation of neurons; positive regulation of cardioblast differentiation; response to hypoxia; epithelial to mesenchymal transition; blood vessel remodeling; negative regulation of cell growth; blood coagulation Disease: Loeys-dietz Syndrome 4
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 by transducing their signal through combinations of transmembrane type I and type II receptors (TGFBR1 and TGFBR2) and their downstream effectors, the SMAD proteins. Disruption of the TGFB/SMAD pathway has been implicated in a variety of human cancers. The encoded protein is secreted and has suppressive effects of interleukin-2 dependent T-cell growth. Translocation t(1;7)(q41;p21) between this gene and HDAC9 is associated with Peters' anomaly, a congenital defect of the anterior chamber of the eye. The knockout mice lacking this gene show perinatal mortality and a wide range of developmental, including cardiac, defects. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Sep 2010]
UniProt Code:	P61812
NCBI GenInfo Identifier:	48429157
NCBI Gene ID:	7042
NCBI Accession:	P61812.1
UniProt Secondary Accession:	P61812,P08112, Q15579, Q15581, Q4VAV9, B4DKC5,
UniProt Related Accession:	P61812
Molecular Weight:	Calculated MW: 47kDa/50kDaObserved MW: 55kDa
NCBI Full Name:	Transforming growth factor beta-2
NCBI Synonym Full Names:	transforming growth factor, beta 2
NCBI Official Symbol:	TGFB2
NCBI Official Synonym Symbols:	LDS4; TGF-beta2
NCBI Protein Information:	transforming growth factor beta-2; G-TSF; cetermin; polyergin; BSC-1 cell growth inhibitor; prepro-transforming growth factor beta-2; glioblastoma-derived T-cell suppressor factor
UniProt Protein Name:	Transforming growth factor beta-2
UniProt Synonym Protein Names:	BSC-1 cell growth inhibitor; Cetermin; Glioblastoma-derived T-cell suppressor factor; G-TSF; PolyerginLatency-associated peptide; LAP
Protein Family:	Transforming growth factor
UniProt Gene Name:	TGFB2
UniProt Entry Name:	TGFB2_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.