Product Name:	Human Transforming growth factor beta-2 (TGFB2) ELISA Kit
Product Code:	HUEB0170
Alias:	Transforming growth factor beta-2, TGF-beta-2, BSC-1 cell growth inhibitor, Cetermin, Glioblastoma-derived T-cell suppressor factor, G-TSF, Polyergin, TGFB2
Uniprot:	P61812
Reactivity:	Human
Range:	15.6-1000 pg/mL
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

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 TGF- beta2 (Transforming Growth Factor beta2) CLIA Kit (HUES00903)	Anti-TGFB2 Antibody (CAB16226)
	Anti-TGFB2 Antibody (CAB3640)
	TGFB2 Antibody (PACO01618)
	TFAP2A Antibody (PACO13633)
	TG Antibody (PACO20669)