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1. D Signosis Human TGF B1 ELISA Catalog Number EA 0208 Introduction The transforming growth factor beta 1 TGF B1 gene codes a multifunctional cytokine that controls proliferation differentiation and other functions in many cell types including cancer cells the surrounding stromal cells immune cells endothelial and smooth muscle cells It causes immunosuppression and angiogenesis which makes the cancer more invasive TGF B also converts effector T cells which normally attack cancer with an inflammatory immune reaction into regulatory suppressor T cells which turn off the inflammatory reaction TGF induces apoptosis in numerous cell types TGF B can act on adipocyte precursor cells 1 TGF 61 has been shown to be a potent inhibitor of the differentiation of adipogenic cell lines 2 In addition a differentiation defective insulin independent cell linederived from the adipogenic cell line 1246 produces in its conditional medium a TGF B1 like polypeptide which could modulate the cell ability to differentiate in an autocrine fashion Increased TGF bl expression was associated with BMI and abdominal adipose tissue in morbid obesity 4 Principle of the assay TGF B1 ELISA is based on the principle of a solid phase enzyme linked immunosorbent assay The assay utilizes a mouse anti human TGF B1 antibody for immobilization on the microtiter wells and chicken anti human TGF f1 antibodies along with streptavidin conjugate2. Drive Suite 10 12 Santa Clara CA 95054 Tel 408 747 0771 Fax 408 864 2182 Reagent preparation before starting experiment Dilute the 5x Assay wash buffer to 1x buffer 40ml 5x Assay wash buffer 160ml ddH20 e Dilute 50 times of human recombinant TGF B1 220ng ml with 1X Diluent buffer to 4400pg ml and then 2 fold serial dilutions Dilute 50 times by adding 4ul Human Recombinant TGF B1 in 200ul 1X Diluent Buffer See Step 2 in Assay Procedure for detailed procedure e Dilute 400 times of biotin labeled chicken anti human TGF B1 antibody with 1X Diluent buffer before use e Dilute 200 times of streptavidin HRP with 1X Diluent buffer before use Assay procedure 1 Cut the sealing film over the plate and remove it from the desired number of well strips Make sure the rest of wells are well sealed 2 See instruction and diagram below for standard preparation Add 200ul 1X Diluent buffer to the 1 well Add 100ul 1X Diluent Buffer to the rest wells of strip Add appropriate amount of protein recombinant follow instruction in Reagent Preparation Mix dilutions in 1 well and transfer 100ul from the 1 well to the next dilution See picture Incubate each well for 1 hr at room temperature with gentle shaking Faget 3 Add 100ul of sample per well and incubate for 1 hour at room temperature with gentle shaking 4 Aspirate each well and wash by adding 200ul of 1X Assay wash buffer Repea
3. d to horseradish peroxidase HRP for detection The test sample is allowed to react simultaneously with the two antibodies resulting in the TGF f 1 molecules being sandwiched between the solid phase and enzyme linked antibodies After incubation the wells are washed to remove unbound labeled antibodies A HRP substrate TMB is added to result in the development of a blue color The color development is then stopped with the addition of Stop Solution changing the color to yellow The concentration of TGF B1 is directly proportional to the color intensity of the test sample Absorbance is measured spectrophotometrically at 450 nm For Research Use Only NANA Plate coated with capture antibody v a _ 4 gt y Add samples Yryryry tke Add biotin labeled detection antibody tt Add streptavidin HRP fet 0o Add substrate of oe oe of of r eseses SELLELE erereereer 2 Itt SF ef OF ef OF Diagram of ELISA Materials provided with the kit 96 well microplate coated with a mouse anti human TGF B1 antibody 4 C Biotin labeled chicken anti human TGF B1 antibodies 20 C Streptavidin HRP conjugate 4 C Recombinant TGF B1 standard 20 C 1X Diluent buffer 4 C 5X Assay wash buffer RT Substrate 4 C Stop Solution 4 C Material required but not provided Microplate reader capable of measuring absorbance at 450 nm Deionized or distilled water Signosis Inc e 1700 Wyatt
4. ica D Morange P et al Plasminogen activator inhibitor 1 transforming growth factor beta 1 and BMI are closely associated in human adipose tissue during morbid obesity Diabetes 2000 49 1374 80 Example of standard curve Human TGF B1 ELISA 100 1000 Human TGF B1 Conc pg ml 10000 a CA 95054 Tel 408 747 0771 Fax 408 864 2182
5. t the process three times for a total of three washes Complete removal of liquid at each wash After the last wash remove any remaining liquid by inverting the plate against clean paper towels 5 Add 100u1 of diluted biotin labeled anti human TGF B1 antibody to each well and incubate for 1 hour at room temperature with gentle shaking 6 Repeat the aspiration wash as in step 4 7 Add 100 ul of diluted streptavidin HRP conjugate to each well and incubate for 45 min at room temperature with gentle shaking 8 Repeat the aspiration wash as in step 4 9 Add 100ul of substrate to each well and incubate for 10 30 minutes 10 Add 50ul of Stop solution to each well The color in the wells should change from blue to yellow 11 Determine the optical density of each well with a microplate reader at 450 nm within 30 minutes References 1 Petruschke T Rohrig K Hauner H Transforming growth factor beta TGFbeta inhibits the differentiation of human adipocyte precursor cells in primary culture Int J Obes Relat Metab Disord 1994 18 532 6 2 Ignotz R and Massague J 1985 Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts Proc Natl Acad Sci USA 82 8530 8540 1985 3 Yamada Y and Serrero G 1989 Characterization of transforming growth factors produced by the insulin independent teratoma derived cell line 1246 3A J Cell Physiol 140 254 263 4 Alessi MC Bastel
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