Our Abpromise guarantee covers the use of ab684 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration. PubMed: 17478566Use at an assay dependent dilution.|
|IHC-Fr||Use at an assay dependent concentration.|
|Flow Cyt||Use 1µg for 106 cells.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
This image is courtesy of an anonymous Abreview
Blocked with 5% milk for 1 hour at 20°C.
Incubated with the primary antibody in 5% milk for 16 hours at 4°C.
The 4 lanes were each loaded with 10ug of protein based upon a BCA assay for protein quantification. However, looking at a subsequent loading control blot (vinvulin) lanes 1 and 4 are under loaded versus lanes 2 and 3.
ab684 staining non-muscle Myosin IIB in WIF-B cells (hepatoma-derived hybrid cell line) by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 1% donkey serum in 1% PBST for 1 hour at 22°C. Samples were incubated with primary antibody (1/50 in blocking buffer) for 3 hours at 22°C. An Alexa Fluor® 488-conjugated donkey anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.
Overlay histogram showing HeLa cells stained with ab684 (red line). The cells were fixed with 80% methanol (5 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. The cells were then incubated with the antibody (ab684, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Triton used under the same conditions.