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Western blot
ELISAAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [3]
- Immunohistochemistry [1]
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- Product number
- 39-8200 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- GAD65 Monoclonal Antibody (ZG002)
- Antibody type
- Monoclonal
- Antigen
- Recombinant full-length protein
- Reactivity
- Human, Mouse, Rat
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- ZG002
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2), Baby Mouse Brain (lane 3) and Mouse Cerebellum (lane 4). The blots were probed with Anti-GAD65 Mouse Monoclonal Antibody (Product # 39-8200, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A 65 kDa band corresponding to GAD65 was observed across tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2), Baby Mouse Brain (lane 3) and Mouse Cerebellum (lane 4). The blots were probed with Anti-GAD65 Mouse Monoclonal Antibody (Product # 39-8200, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A 65 kDa band corresponding to GAD65 was observed across tissues tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-GAD65 Monoclonal Antibody (ZG002) (Product # 39-8200) and a 60 kDa band corresponding to GAD65 was observed across tissues tested except Mouse and Rat Kidney which are reported to be negative. An additional band at ~25 kDa (*) corresponding to Mouse IgG was alos observed. Whole cell extracts (30 µg lysate) of Mouse brain (Lane 1), Rat brain (Lane 2), Mouse kidney (Lane 3), and Rat kidney (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blots were probed with the primary antibody (0.5 µg/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of GAD2/GAD65 showing staining in the cytoplasm and membrane of paraffin-embedded human pancreatic tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a GAD2/GAD65 mouse monoclonal antibody (Product # 39-8200) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
