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Synthetic peptide corresponding to amino acids 2 - 15 (DDDIAALVVDNGSG) of Human beta Actin cytoplasmic domain (slightly modified)
Our Abpromise guarantee covers the use of ab92436 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ELISA||Use at an assay dependent concentration.|
|Dot blot||Use at an assay dependent concentration.|
|WB||1/1000 - 1/3000. Predicted molecular weight: 42 kDa.|
|Flow Cyt||Use 0.1-1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.|
|ICC/IF||Use a concentration of 10 µg/ml.|
|IP||Use a concentration of 5 µg/ml.|
beta Actin was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to beta Actin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab92436.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.
Band: 42kDa; beta Actin
ICC/IF image of ab92436 stained HeLa cells. The cells were 100% methanol fixed (5 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 ab92436 at 10 µg/mL overnight at +4°C. The secondary antibody (green) was a goat anti-mouse DyLight® 488 (IgG; H+L) (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.
Overlay histogram showing HeLa cells stained with ab92436 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween 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 (ab92436, 0.1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse Alexa Fluor® 488 (IgG; H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
ab92436 has not yet been referenced specifically in any publications.