Immunofluorescence analysis of Troponin I was done on Primary Human Cardiomyocytes. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ABfinity™ Troponin I Recombinant Rabbit Monoclonal Antibody (701585) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing striated muscle fiber localization. Panel e is a no primary antibody control. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||human Troponin I|
|Contains||0.09% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||Assay Dependent|
|Immunofluorescence (IF)||Assay Dependent|
|Immunohistochemistry (IHC)||Assay Dependent|
|Western Blot (WB)||Assay Dependent|
* 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.
Troponin and tropomyosin are the main proteins responsible for calcium regulation of actin-myosin interactions. Tropomyosin is a rod-shaped molecule that binds in the groove of the actin helix, and prevents the attachment of the myosin fiber heads. The troponin complex consists of three polypeptide subunits, troponin T, troponin I, and troponin C. Troponin T (for tropomyosin binding unit) attaches the troponin complex to tropomyosin which results in tropomyosin being situated in the myosin-binding site on the actin molecule. Troponin I (for inhibitory subunit) binds to actin and troponin T, and this complex inhibits actin-myosin binding even in the presence of calcium. Troponin C (for calcium-binding subunit) binds to troponin T and troponin I, and makes the complex dependent upon calcium. With calcium present, troponin C binds up to 4 molecules and changes the position of the troponin and tropomyosin complex, so that myosin is able to bind to the actin filament. Troponin I is present in three different isoforms in mammals, a slow-twitch skeletal muscle-specific form, a fast-twitch muscle-specific form, and a cardiac muscle-specific form.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.