Immunofluorescence analysis of SMAD-5 was done on 70% confluent log phase HeLa cells. 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 SMAD5 Antibody (ZS004)(395700) at 1µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) at a dilution of 1:400 for 45 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 cytoplasmic and Nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Published species reactivity||Mouse|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||Recombinant protein derived from an internal region of the human and rat Smad5 proteins|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:100|
|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.
|Western Blot (WB)||See 1 publications below|
SMADs are members of the MAD-related family of molecules. MAD-related proteins are a family of intracellular proteins that are essential components in the signaling pathways of the serine/threonine kinase receptors of the transforming growth factor beta superfamily (1). SMADs can be divided into receptor-regulated SMADs (R-SMADs: SMAD1, 2, 5, 8 and 9), common-mediator SMAD (co-SMAD: SMAD4), and inhibitory SMADs (I-SMADs: SMAD6 and 7). SMAD1, 5, 8 and 9 have high degrees of homology and antibodies are available that recognize sequences common to all of them. SMAD8 and SMAD9 are typically used as alternate names for one another in the literature.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment.
39-5700 was used in western blot to determine if Smad1 suppresses hematopoiesis.
|Cook BD,Liu S,Evans T||Blood (117:6489)||2011|