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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human alpha Tubulin.
(Peptide available as ab23537.)
Our Abpromise guarantee covers the use of ab89984 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IF||Use a concentration of 5 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 53 kDa (predicted molecular weight: 50 kDa).Can be blocked with Human alpha Tubulin peptide (ab23537).|
|ICC/IF||Use a concentration of 5 µg/ml.|
ab89984 staining alpha Tubulin in Caco-2 cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab89984 at 5μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Chicken secondary (ab150173) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab89984 overnight at 4°C. Antibody binding was detected using ab175787 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
ICC/IF image of ab89984 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab89984 at 5ug overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti- chicken IgY (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in HepG2 PFA fixed cell types at 5ug/ml.
Western blot image using the Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-20%) (ab119220) with the
Prism Ultra Protein Ladder (ab116028) 5µl used. We recommend using our ECL substrate kit (ab65623) .
20ug of Lysate per lane and detection using ab89984 diluted to 1ug/ml.
Lane 1: Hela cell lysate
Lane 2: Jurkat cell lysate
Lane 3: A431 cell lysate
Lane 4: HEK293 cell lysate
Lane 5: HepG2 cell lysate
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"