Sample Preparation of Transforming Growth Factor Beta (TGFβ)

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Transforming growth factor beta (TGF-β) is a group of transforming growth factor beta superfamily of cytokines, which can control proliferation, cellular differentiation, and other functions in most cells. It is widely located in normal cells and transformed cells. It is a type of cytokine which plays a role in promotion of embryonic development, immunomodulatory effect, growth suppression, wound healing, angiogenesis, extracellular matrix precipitation and so on. This cytokine can promote the transformation of normal fibroblasts from adherent growth to suspension. Therefore, it is named.

TGF-β exists in Mammals at least three isoforms called TGF-β1, TGF-β2 and TGF-β3. The genes of TGF-βs are located in different chromosomes and the amino acide homologies among them range from 70-80%. Among these TGF-βs, the proportion of TGF-β1 is more than 90%. And TGF-β1 is secreted by many cell types including macrophages, lymphocytes, endothelial cells and chondrocytes. It also can be secreted by leukemia cells, mainly distributed in human platelets and mammal bones. While TGF-β2 is secreted by Keratinocytes, granule cell layer and neuroglioma cells. Moreover, TGF-β3 is derived from Mesenchymal cell.

TGF-βs are secreted by many cell types. All three TGF-βs are synthesized as disulfide-linked homodimers containing a propeptide region. After it is synthesized, the TGF-β homodimer interacts with a Latency Associated Peptide (LAP, an N-terminal signal peptide of 20-30 amino acids derived from the TGF beta gene product) forming a complex called Small Latent Complex (SLC). This complex remains in the cell until it is bound by another protein called Latent TGF-β-Binding Protein (LTBP), forming a larger complex called Large Latent Complex (LLC). It is LLC that get secreted to the extracellular matrix (ECM). That is to say, most TFG-β is secreted as a latent form(LLC) in biological samples. The sample is needed to be further processed in order to release TGF-β from LLC. After preparation, the TGF-β level in samples can be accurately measured since the antibodies in the kits is specifically against to the free TGF-β. The sample preparation procedure is listed below.

[ SAMPLE Preparation PROCEDURE ]

Serum/Plasma

1. To 50μL of serum/plasma, add 10μL of 1 N HCl. Mix well.

2. Incubate 10 minutes at room temperature.

3. Neutralize the acidified sample by adding 10μL of 1.2 N NaOH/0.5 M HEPES. Mix well. Add 80μL Standard Diluent. Mix well. Assay immediately.

4. The concentration read off the standard curve must be multiplied by the appropriate dilution factor, 3.

Cell culture supernates

1. To 100μL of cell culture supernate, add 20μL of 1 N HCI. Mix well.

2. Incubate 10 minutes at room temperature.

3. Neutralize the acidified sample by adding 20μL of 1.2 N NaOH/0.5 M HEPES. Mix well. Assay immediately.

4. The concentration read off the standard curve must be multiplied by the dilution factor, 1.4.

Take ELISA Kit for TGF-β1 (SEA124Hu) as a example, we used it to measure the TGF-β1 levels in samples before and after treatment and made a comparison. As shown in figure 1, the TGF-β1 levels in serum samples after treatment are increased. It is because that the TGF-β1 is released from LLC(the complex of TGF-β1, LAP and LTBP) after treatment. The data may be not precise by directly measure the TGF-β1 levels in samples without treatment. In order to obtain the accurate quantitative measurement of TGF-β1, pre-treatment in samples is recommended before test.

Sample Preparation of Transforming Growth Factor Beta (TGFβ)

 

Figure1 The comparison of TGF-β1 level in samples: before and after treatment

The references related to the products of TGF-βs are shown in Table1. Your attention is invited.

Transforming Growth Factor Beta 1 (TGFb1)
Catalog  Reference
SEA124Hu Evidence for the complementary and synergistic effects of the three-alkaloid co mbination regimen containing berberine, hypaconitine and skimmianine on the ulcerative colitis rats induced by trinitrobenzene-sulfonic acid
Biomarkers of Fibroproliferative Healing in Fibrosing Idiopathic Interstitial Pn eumonias
A Combinatorial Relative Mass Value Evaluation of Endogenous Bioactive Pro teins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Inter vertebral Discs: Identification of Potential Target Proteins for Gene Therapeuti c Approaches
SEA124Mu Metformin attenuates cardiac fibrosis by inhibiting the TGFβ1–Smad3 signalli ng pathway
SEA124Ra Incorporating pTGF-β1/calcium phosphate nanoparticles with fibronectin into 3-dimensional collagen/chitosan scaffolds: efficient, sustained gene delivery to stem cells for chondrogenic differentiation.
A Soluble Antigen-Specific Factor from CD4+CD25+ T Cells of OVA Toleran t Mice Inducing OVA-Specific Peripheral Tolerance Independently in vivo
The Intervention Effect of Rosiglitozone in Ovarian Fibrosis of PCOS Rats
Fibrogenic response of hepatic stellate cells in ovarectomised rats exposed to k etogenic diet
Multitargeted protective effect of Abacopteris penangiana against carrageenaninduced chronic prostatitis in rats.
SEA124Gu  
SEA124Rb Chronic angiotensin-(1–7) administration improves vascular remodeling after a ngioplasty through the regulation of the TGF-β/Smad signaling pathway in rab bits
SEA124Si  
SEA124Ca Increased circulating concentrations of PDGF-BB and TGF-β1 in canine gener alised demodicosis
SEA124Po  
SEA124Bo EVALUATION OF PHYSICO-CHEMICAL PROPERTIES OF COLOSTRU M SUPPLEMENTED DAHI
SEA124Eq  
SEA124Ga  
Transforming Growth Factor 
Catalog Reference
CEA218Hu  
SEA218Mu  
CEA218Mu  
SEA218Ra  
SEA218Si  
SEA218Ca  
SEA218Bo EVALUATION OF PHYSICO-CHEMICAL PROPERTIES OF COLOSTRU M SUPPLEMENTED DAHI
Transforming Growth Factor Beta 3 (TGFb3)
Catalog Reference
SEB949Hu A Combinatorial Relative Mass Value Evaluation of Endogenous Bioactive Pro teins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Inter vertebral Discs: Identification of Potential Target Proteins for Gene Therapeuti c Approaches
SEB949Mu  
SEB949Ra  
SEB949Po  

Table 1 References of ELISA Kits for TGF-βs Detection

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