Immunofluorescence analysis of SMAD2 [pT8] was done on 70% confluent log phase TGF-beta treated HeLa cells (serum starved for 16 hours followed by treatment with 20 ng/ml TGF-beta for 1 hour). 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 [pT8] Recombinant Rabbit Monoclonal Antibody (700050) at 1µg/mL 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 [pT8] and panel e is a no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||3µg|
|Flow Cytometry (Flow)||1-3µg/10^6 cells|
|Western Blot (WB)||1-3µg/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.
This antibody is predicted to react with bovine, Drosophila, mouse, rat and Xenopus based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. 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™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. 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.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
SMAD2, also known as MADH2 or MAD2 regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. Smad2 interacts with the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation into the nucleus is a central event in TGF beta signaling. Phosphorylation of threonine 8 in the calmodulin-binding region of the MH1 domain by extracellular signalregulated kinase 1 (ERK1) enhances Smad2 transcriptional activity, which is negatively regulated by calmodulin.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.