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Full length native protein (purified) of Chicken alpha Tubulin (extracted from brain).
Excellent as a protein loading control antibody.
Our Abpromise guarantee covers the use of ab7291 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 1µg for 106 cells.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|IP||Use at an assay dependent dilution.|
|IHC-P||Use at an assay dependent dilution. Antigen retrieval is not essential but may optimise staining.|
|IHC-Fr||Use at an assay dependent concentration.|
|Electron Microscopy||Use at an assay dependent dilution.|
|WB||1/5000 - 1/10000. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa). Lower background can be obtained by diluting ab7291 to 1:10000 and incubating overnight at 4C (Customer communication).This antibody is ideal for use as a Western blot loading control.Works under both reducing and non-reducing conditions.|
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 2% Bovine Serum Albumin before being incubated with ab7291 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406
ICC/IF image of ab7291 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab7291, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
Immunofluorescent imaging of human cells (U2OS) with ab7291 reveals a delicate network of alpha-tubulin (green) located exclusively in the cytoplasm. The nucleus is stained blue.
IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes. All blocking and incubation steps carried out at 37 degrees.
Unfortunately, due to size constraints for images on our website, we are unable to show the full uncompressed picture in all its glory!
ab7291 at 1/400 staining MCF-10A cells (human mammary epithelial cell line) by ICC/IF. The cells were methanol fixed, blocked with BSA and incubated with the antibody for 16 hours. An Alexa-Fluor ® 488 conjugated goat anti-mouse antibody was used as the secondary. The image shows alpha tubulin staining in green and DAPI counterstain in blue.
Western blot using ab7291 on Hela Whole Cell Lysate (20µg/lane).
Exposure: 10 sec.
Lane 1: ab7291 at 1/5000
Lane 2: ab7291 at 1/10000.
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