Western blot analysis was performed on membrane enriched extracts (30 ug lysate) of SH-SY5Y (Lane 1) and SH-SY5Y treated with Hydrogen Peroxide (1 mM Hydrogen Peroxide for 30 minutes). The blot was probed with Anti-GAPDH Mouse Monoclonal Antibody (Product # LF-MA0100, 2 ug/ml) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 ug/ml, 1:2500 dilution). A 37 kDa band corresponding to GAPDH was observed upon treatment with Hydrogen Peroxide. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Sulfonylated peptide, KLH coupled, corresponding to the oxidation sensitive site sequence to human GAPDH protein.|
|Storage buffer||HEPES with 0.15M NaCl, 0.01% BSA, 50% glycerol|
|Contains||0.03% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||0.5-2 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
A suggested positive control for this product is HeLa cells treated with H2O2.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a catalytic enzyme commonly known to be involved in glycolysis. The enzyme exists as a tetramer of identical 37-kDa subunits. GAPDH catalyzes the reversible reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphophate in the presence of NADPH. Apart from playing a key role in glycolysis, this ubiquitously expressed enzyme also displays other activities unrelated to its glycolytic function. GAPDH is reported to be involved in the processes of DNA replication , DNA repair , nuclear RNA export, membrane fusion and microtubule bundling. Other studies also provide evidence of GAPDH playing an essential part of the program of gene expression observed in apoptosis and as part of the cellular phenotype of age-related neurodegenerative diseases.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.