Immunofluorescence analysis of SMAD2 [pSpS465/467] was done on 70% confluent log phase HepG2 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ABfinity™ SMAD2 [pSpS465/467] Recombinant Rabbit Oligoclonal Antibody (710612) at a dilution of 1:200 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 nuclear localization of SMAD2 [pSpS465/467]. Panel e shows competition with SMAD2 [pSpS465/467] peptide. Panel f is no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to human SMAD2, aa 461-467, phosphorylated at Serine 465/467|
|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)||3-5 µg/ml|
|Immunofluorescence (IF)||3-5 µg/ml|
|Western Blot (WB)||1-2 µg/ml|
* 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.
ABfinity™ recombinant oligoclonal antibodies are rabbit antibody pools generated from isolated DNA libraries. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ oligoclonal antibodies recognize multiple epitopes and therefore exhibit greater sensitivity and because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
This antibody is predicted to react with mouse, bovine, zebrafish and drosophila based on sequence homology.
Smad2 belongs to SMAD family of proteins, which are signal transducers and transcriptional modulators that regulate multiple signaling pathways. Smad2 mediates transforming growth factor (TGF)-beta signaling and regulates physiological processes such as cell proliferation, apoptosis and differentiation. In response to the TGF beta signal, Smad2 undergoes phosphorylation, which then interacts with Smad4 and enters into the cell nucleus to bind to various promoter elements to regulate expression of multiple genes. Phosphorylation of Smad2 is key for its function, especially to associate with Smad4 and to get entry into the cell nucleus.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.