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Chicken gizzard actin.
Our Abpromise guarantee covers the use of ab3280 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 1-2µg for 106 cells.|
|IP||Use at 2 µg/mg of lysate. Use Protein G. This has been tested in IP on "denatured" cell lysate (treated with 5% SDS).|
|WB||Use a concentration of 0.5 µg/ml. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa).|
|IHC-P||Use a concentration of 0.5 - 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ICC/IF||Use at an assay dependent concentration. See Bryce et al. Fix cells with 4% paraformaldehyde and permeabilize with chilled methanol.|
Lanes: 1-3: 4oC (x1 freeze/thaw)
Lanes: 4-6: 4oC (x2 freeze/thaws)
Lanes 7-9: 4oC (x4 freeze/thaws)
This image is courtesy of an anonymous AbreviewBlocking Step: 5% Milk for 30 minutes at 25°C.
ab3280 (1µg/ml) staining actin in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining throughout the tissue.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
ICC/IF image of ab3280 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 (ab3280, 5µg/ml) overnight at +4°C. The secondary antibody (green) was a goat anti-mouse DyLight® 488 (IgG - H&L, pre-adsorbed) (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 ab3280 (red line). The cells were fixed with 80% methanol (5 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 (ab3280, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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