TSH Receptor Monoclonal Antibody (49) Catalog Number MA3-218 Product data sheet

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1 Website: thermofisher.com Customer Service (US): ext. 1 Technical Support (US): ext. 441 TSH Receptor Monoclonal Antibody (49) Catalog Number MA3-218 Product data sheet Details Size 100 µl Host/Isotope Class Type Clone 49 Immunogen Conjugate Form Storage buffer Contains Storage Conditions Mouse / IgG1 Monoclonal Antibody Purified extracellular domain of recombinant human thyrotropin receptor. Unconjugated Liquid ascites 0.05% sodium azide -20 C, Avoid Freeze/Thaw Cycles Species Reactivity Tested species reactivity Human, Pig Published species reactivity Not Applicable Tested Applications Dilution * ELISA (ELISA) Assay dependent Immunocytochemistry (ICC) 1:250 Immunofluorescence (IF) 1:250 Western Blot (WB) 1:250 Published Applications Miscellaneous PubMed (MISC) See 3 publications below * Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls. Product specific information MA3-218 detects both glycosylated and nonglycosylated thyroid stimulating hormone receptor (TSHR) from porcine tissues as well as recombinant human TSHR. MA3-218 has been successfully used in Western blot, immunofluorescence, and ELISA procedures. By Western blot, this antibody detects a 50 kda and a 63 kda protein representing unglycosylated and glycosylated TSHR respectively, in transfected Sf9 cell extract. By ELISA MA3-218 has been used in TSH competitive inhibition studies. The MA3-218 antigen is purified extracellular domain of recombinant human thyrotropin receptor. This antibody recognizes an epitope between amino acid residues of human TSHR. Background/Target Information The thyroid stimulating hormone (thyrotropin) receptor (TSHR) is expressed on the surface of thyrocyte cell membranes and is crucial for thyroid gland growth and function. TSHR and quote;s are targets for the pathogenesis of many autoimmune diseases including Graves and quote; disease and primary myxedema. This 7-transmembrane-domain protein contains 5 potential extracellular N-linked glycosylation sites. Antibodies to TSHR can affect receptor function in a number of different ways. TSHR antibodies which can inhibit TSH binding to the receptor are called TSH binding inhibitor immunoglobulins or TBIIs.

2 Product Images For TSH Receptor Monoclonal Antibody (49) TSH Receptor Antibody (MA3-218) in IF Immunofluorescence analysis of Thyrotropin Receptor was performed using 70% confluent log phase A431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with TSH Receptor (49) Mouse Monoclonal Antibody (Product # MA3-218) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1: 300). Panel d represents the merged image showing membranous localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

3 TSH Receptor Antibody (MA3-218) in IF Immunofluorescent analysis of Thyrotropin Receptor in A431 cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Thyrotropin Receptor monoclonal antibody (Product # MA3-218) at a dilution of 1:200 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Thyrotropin Receptor staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification. TSH Receptor Antibody (MA3-218) in WB

4 Western blot analysis was performed on membrane-enriched extracts (30 µg lysate) of A-431 (Lane 1), IMR- 32 (Lane 2) and Jurkat (Lane 3). The blots were probed with Anti- TSH Receptor Mouse Monoclonal Antibody (Product # MA3-218, 1: 250 dilution) and detected by chemiluminescence using Goat anti-mouse IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/ml, 1:4000 dilution). A 63 kda band corresponding to TSH Receptor was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex NuPAGE 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock Electrophoresis System (Product # EI0002), and Novex Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iblot 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

5

6 PubMed References For TSH Receptor Monoclonal Antibody (49) 3 Miscellaneous PubMed References Species / Dilution Summary MA3-218 was used in western blot and immunoprecipitation to study the expression and function of thyrotropin receptor in murine dendritic cells and in lymph node T cells. Not Applicable / Not Cited Not Applicable / Not Cited Not Applicable / Not Cited Journal of immunology (Baltimore, Md. : 1950) (Jun 2000; 164: 6158) "The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RBhigh lymph node T cells: functional role for thyroid-stimulating hormone during immune activation." Author(s):Bariaçik EU,Klein JR PubMed Article URL: MA3-218 was used in ELISA to investigate the expression of met-gp and characterize the difference of epitopes between met-gp and ETSHr-gp Endocrinology (Apr 1997; 138: 1559) "Differential reactivities of recombinant glycosylated ectodomains of mouse and human thyrotropin receptors with patient autoantibodies." Author(s):Patibandla SA,Seetharamaiah GS,Dallas JS,Thotakura NR,Peake RL,Prabhakar BS PubMed Article URL: MA3-218 was used in blocking/activating experiment and ELISA to characterize human TSH receptor antibodies in vitro and in vivo Endocrinology (Jul 1995; 136: 2817) "Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: antibodies can bind to discrete conformational or linear epitopes and block TSH binding." Author(s):Seetharamaiah GS,Wagle NM,Morris JC,Prabhakar BS PubMed Article URL:

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