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Full length native protein (purified) corresponding to Pig alpha Tubulin aa 37-154.
Our Abpromise guarantee covers the use of ab7750 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. Ab81216-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.|
|IHC-P||Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ELISA||Use at an assay dependent concentration.|
|ICC||1/100. For ICC, we recommend not only fixing but also permeabilizing the cells.|
|ICC/IF||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 - 2 µg/ml. Detects a band of approximately 55 kDa.|
This image is courtesy of an Abreview submitted by Dr sudhish mishra
ICC/IF image of ab7750 stained HeLa cells. The cells were 4% formaldehyde 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 (ab7750, 5µg/ml) overnight at +4°C. The secondary antibody (green) was anti-mouse DyLight® 488 (ab96879) used at a 1/250 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.
Ab7750 staining human normal skin. Staining is localised to the cytoplasm.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved with a retrieval buffer EDTA pH 9.0 . Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with an amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
Overlay histogram showing HEK293 cells stained with ab7750 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab7750, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was anti-mouse DyLight® 488 (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Triton used under the same conditions.
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