Immunofluorescence analysis of beta-3 Tubulin 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 beta-3 Tubulin (2G10) Mouse Monoclonal Antibody (MA1118X) at 2µg/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A28175) 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 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||Bovine, Guinea pig, Hamster, Human, Mouse, Pig, Rabbit, Rat|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||A synthetic peptide corresponding to amino acids 436-450 from rat neuronal specific beta-3 tubulin.|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:50|
|Immunohistochemistry (IHC)||1:50 - 1:200|
|Immunoprecipitation (IP)||5 µg|
|Western Blot (WB)||1:500 - 1:2000|
* 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 analysis of MA1-118 detects an ~50 kDa protein in neuronal-type cells. In bovine, a unknown band at ~32 kDa is also detected. MA1-118 shows specificity to beta-3 Tubulin and is non-reactive to lysates from non-neuronal cell types (e.g. HeLa cell lysate).
Beta tubulins are one of two core protein families (alpha and beta tubulins) that heterodimerize and assemble to form microtubules. Beta-III tubulin is primarily expressed in neurons and may be involved in neurogenesis, axon guidance and maintenance. Mutations in this gene are the cause of congenital fibrosis of the extraocular muscles type 3. Beta-III tubulin was also detected in Sertoli cells of the testis and transiently in non-neuronal embryonic tissues. Glutamate residues at the C-terminus of beta III tubulin can be glutamylated. The precise function of such modifications is unclear. Tubulin is phosphorylated on Ser-172 by CDK1 during cell cycle progression. Ser-172 phosphorylation inhibits tubulin incorporation into microtubules.
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