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ab26116 mouse monoclonal [K-87] to GAD67 specically recognizes GAD67 and has no cross reactivity with GAD65; this has been shown on western blots of mouse brain or purified recombinant GAD67 and GAD65. In immunohistochemical analysis of brain sections, the K-87 monoclonal recognizes GAD67 in nerve cell bodies and has an enhanced ability to detect GAD67 in dendrites and axon buttons compared to the original anti-GAD67 K-2 polyclonal antibody (see Kaufman et al, 1991).
Our Abpromise guarantee covers the use of ab26116 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 10 µg/ml.|
|Flow Cyt||Use 1µg for 106 cells.
(methanol fixed cells)
|IHC-P||1/1000 - 1/10000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. An antigen retrieval step is necessary (e.g. incubating the sections at 90 degrees C in a 50mM citrate buffer). Cell body labeling is optimized when Triton is omitted from the tissue processing. Axon terminal labeling is substantially increased (and cell body labeling decreased) when Triton is included (see Soghomonian et al, 1998).|
|WB||1/1000 - 1/20000. Detects a band of approximately 67 kDa (predicted molecular weight: 67 kDa).|
Immunohistochemical staining of mouse pancreatic islet beta cells after antigen retrieval with ab26116 at a dilution of 1/1000.
ICC/IF image of ab26116 stained PC12 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 (ab26116, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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 PC12 cells stained with ab26116 (red line). The cells were fixed with methanol (5 min) and 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 (ab26116, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (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 slightly decreased signal in PC12 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.
Please note that Abcam does not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.