Anti-GAD65/GAD2 Antibody Picoband™ (monoclonal, 7G2)

Figure 1. Western blot analysis of GAD65/GAD2 using anti-GAD65/GAD2 antibody (M03142).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat brain tissue lysates,
Lane 2: mouse brain tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-GAD65/GAD2 antigen affinity purified monoclonal antibody (Catalog # M03142) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for GAD65/GAD2 at approximately 62KD. The expected band size for GAD65/GAD2 is at 62KD.

Figure 2. IHC analysis of GAD65/GAD2 using anti-GAD65/GAD2 antibody (M03142).
GAD65/GAD2 was detected in paraffin-embedded section of human glioma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-GAD65/GAD2 Antibody (M03142) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

Figure 3. IF analysis of GAD65/GAD2 using anti- GAD65/GAD2 antibody (M03142).
GAD65/GAD2 was detected in immunocytochemical section of Hela cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 4μg/mL mouse anti- GAD65/GAD2 Antibody (M03142) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Figure 4. Flow Cytometry analysis of U20S cells using anti- GAD65/GAD2 antibody (M03142).
Overlay histogram showing U20S cells stained with M03142 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-GAD65/GAD2 Antibody (M03142, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 5. Flow Cytometry analysis of 293T cells using anti- GAD65/GAD2 antibody (M03142).
Overlay histogram showing 293T cells stained with M03142 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-GAD65/GAD2 Antibody (M03142, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.