Immunofluorescent analysis of Ecm29 was performed using 70% confluent log phase HeLa 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 Ecm29 Rabbit Polyclonal Antibody (PA3-035) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) 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 (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to the C-terminal residues R(1821) P E L Q E K A A L L K K T L E N L E(1839) of human Ecm29.|
|Storage buffer||whole serum|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:250|
* 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.
|Immunohistochemistry (IHC)||See 1 publications below|
PA3-035 detects Ecm29 in human and mouse samples.
PA3-035 has been used successfully in Western blot and immunofluorescence procedures. In Western blot analysis of HeLa cell extracts this antibody detects a ~205 kDa protein representing Ecm29.
The PA3-035 immunogen is a synthetic peptide corresponding to the C-terminal residues R(1821) P E L Q E K A A L L K K T L E N L E(1839) of human Ecm29.
Ecm29 appears to be one of three proteins which associate significantly with proteasomes. Ecm29 has been shown to enhance the stability of the proteasome. Ecm29 deletion has been shown to produce 26S proteasomes which dissociated in the absence of ATP. It was thus suggested that Ecm29 tethers the regulatory complex to the proteasome.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles.
PA3-035 was used in immunohistochemistry to investigate the role of mammalian ecm29 in the 26S proteasome
|Gorbea C,Goellner GM,Teter K,Holmes RK,Rechsteiner M||The Journal of biological chemistry (279:54849)||2004|