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Full length native protein from human erythrocytes.
Alternative versions available:
Our Abpromise guarantee covers the use of ab9484 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 a concentration of 5 µg/ml.|
|WB||Use a concentration of 0.1 - 1 µg/ml. Detects a band of approximately 36 kDa (predicted molecular weight: 40.2 kDa).
NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have thoroughly investigated the blocking conditions for this antibody following concerning customer feedback on the lack of signal with some of the vials. We found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see images). This change in the characteristics of the antibody is due to a recent update to the production process.
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|Flow Cyt||Use 1µg for 106 cells.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Western blot using ab9484 at 1/5000.
Lane 1: Hela whole cell (Human)
Lane 2: 3T3 cell (Mouse)
Lane 3: Rat brain
Lane 4: Xenopus embryo
Lane 5: Chicken Liver
Lane 6: EBTr cell (Cow)
Lane 7: CHO cell (Chinese hamster)
Lane 8: Pig liver
Secondary ab: Rabbit polyclonal to mouse IgG (ab6728 at 1/5000). Exposure time: 10 seconds.
The membrane was blocked in 5%BSA in TBST for one hour, then incubated for one hour in primary antibody diluted in TBST.
ICC/IF image of ab9484 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 (ab9484, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, anti mouse-DyLight® 488 (IgG H+L) 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 ab9484 (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 (ab9484, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was 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 IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
ab9484 staining GAPDH in A549 lung cancer cells by Flow Cytometry. Cells were harvested in trypsin, fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100. The sample was incubated with the primary antibody (1/100 in 1x HBSS + 0.02% Triton X-100 + 1.5% FBS) for 3 hours at 25°C. An Alexa Fluor® 488-conjugated goat anti-mouse IgG polyclonal (1/2000) was used as the secondary antibody.
Gating Strategy: No gating.
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