Immunofluorescence analysis of alpha Tubulin was performed using 70% confluent log phase NIH/3T3 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 Alpha Tubulin Rabbit Polyclonal Antibody (PA120988) 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 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||Amphibian, Bovine, Dog, Gerbil, Guinea pig, Human, Mouse, Non-human primate, Pig, Rat, Xenopus|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to the residues M(426) A A L E K D Y E E V G V D S V E G E G E E E G E E(450) of human alpha Tubulin.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7.4, with 1% BSA|
|Contains||0.09% sodium azide|
|Storage Conditions||4° C, do not freeze|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:200|
|Immunoprecipitation (IP)||Assay dependent|
|Western Blot (WB)||1 - 3 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.
PA1-20988 detects apha Tubulin in human, mouse, rat, Xenopus laevis, bovine, guinea pig, gerbil, and porcine samples. Expected to cross react with newt (100% identity with immunogen) due to sequence homology.
PA1-20988 has been successfully used in immunofluorescence, immunohistochemistry (FFPE), immunoprecipitation and Western blot procedures. By Western blot, PA1-20988 detects a ~55 kDa band representing alpha Tubulin. Formalin-fixed paraffin embedded tissue sections require boiling in 10 mM citrate buffer (pH 6.0) prior to immunostaining. Positive control of lung tissue slides suggested.
The PA1-20988 immunogen is a synthetic peptide corresponding to the residues M(426) A A L E K D Y E E V G V D S V E G E G E E E G E E(450) of human alpha Tubulin. This sequence is 100% conserved in newt alpha Tubulin.
Fig. 1 shows specific staining of paraffin embedded human lung sections using PA1-20988.
Tubulin is the major building block of microtubules. This intracellular cylindrical filamentous structure is present in almost all eukaryotic cells. Microtubules function as structural and mobile elements in mitosis, intracellular transport, flagellar movement and in the cytoskeleton. Tubulin is a heterodimer which consists of alpha tubulin and beta tubulin.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.