Immunofluorescence analysis of Ubiquitin was performed using 70% confluent log phase SH-SY5Y 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 Ubiquitin Rabbit Polyclonal Antibody (PA5-16829) at 2ug/ml 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) at 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant full-length human ubiquitin protein|
|Storage buffer||PBS, pH 7.6, with 0.2% BSA|
|Contains||15mM sodium azide|
|Storage Conditions||4° C|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50-1:200|
* 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.
PA5-16829 targets Ubiquitin in IHC (P) applications and shows reactivity with Human samples.
The PA5-16829 immunogen is recombinant full-length human ubiquitin protein.
Ubiquitin (Ub) plays a very important role in regulated non-lysosomal ATP dependent protein degradation. The protein to be degraded is conjugated to Ub and the ubiquinated protein is then selectively degraded by a 26S complex, multicatalytic cytosolic and nuclear protease termed proteasome. The Ub-proteasome proteolytic pathway, which is a complex process, is implicated to be of great importance for regulating numerous cellular processes.
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