The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 40 kDa (predicted molecular weight: 40 kDa).
Use a concentration of 5 µg/ml.
FunctionHas both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. Participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis. Nuclear functions are probably due to the nitrosylase activity that mediates cysteine S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2 and PRKDC (By similarity). Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate.
PathwayCarbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.
Sequence similaritiesBelongs to the glyceraldehyde-3-phosphate dehydrogenase family.
Post-translational modificationsS-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus. ISGylated.
Cellular localizationCytoplasm > cytosol. Nucleus. Cytoplasm > perinuclear region. Membrane. Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions.
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody - Loading Control (ab15822)
ab15822 staining GAPDH in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab15822 at 5μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Chicken secondary (ab150173) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
Western blot - GAPDH antibody (ab15822)
Anti-GAPDH antibody - Loading Control (ab15822) at 1 µg/ml + HeLa whole cell lysate at 20 µg
Secondary Goat Anti-Chicken IgG (H&L) secondary at 1/10000 dilution
Performed under reducing conditions.
Predicted band size : 40 kDa
ab15822 detects a band of ~ 40 kDa in HeLa, Jurkat and A431 whole cell lysates.
ICC/IF image of ab15822 stained human MCF7 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab15822, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-chicken IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, HEK 293 and HepG2 cells.
References for Anti-GAPDH antibody - Loading Control (ab15822)
This product has been referenced in:
Kelly R & Davey SK Tousled-like kinase-dependent phosphorylation of Rad9 plays a role in cell cycle progression and G2/M checkpoint exit. PLoS One8:e85859 (2013).
Read more (PubMed: 24376897) »