Tested applicationsSuitable for:
ICC/IF, IHC-P, WBmore details Unsuitable for:
Mouse, Human Predicted to work with:
Rat, Chicken, Guinea pig, Turkey, Pig, Xenopus laevis, Chimpanzee, Reptiles, Rhesus monkey, Gorilla
Synthetic peptide, corresponding to a region between residues 250-300 of Human GAPDH. (NP_002037.2).
Whole cell lysate from HeLa and mouse NIH3T3 cells.
This antibody gave a positive result in IHC in the following FFPE tissue: Human normal skeletal muscle.
This antibody gave a positive result when used in the following formaldehyde fixed cell lines: HepG2.
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/2000 - 1/10000. Detects a band of approximately 38 kDa (predicted molecular weight: 36 kDa).
Application notesIs unsuitable for IP.
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma (left) and mouse squamous cell carcinoma (right) tissues labelling GAPDH with ab70699 at 1/200 (1µg/ml). Detection: DAB.
ab70699 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 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 ab70699 at 5µg/ml overnight at +4°C. The secondary antibody (green) was anti-Rabbit DyLight® 488 (ab96899) IgG (H+L) used at a 1/250 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.
Western blot - GAPDH antibody (ab70699)
All lanes : Anti-GAPDH antibody (ab70699) at 0.04 µg/ml
Lane 1 : Whole cell lysate from HeLa cells at 50 µg Lane 2 : Whole cell lysate from HeLa cells at 15 µg Lane 3 : Whole cell lysate from HeLa cells at 5 µg Lane 4 : Whole cell lysate from NIH 3T3 cells at 50 µg
IHC image of GAPDH staining in Human skeletal muscle formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab70699, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
References for Anti-GAPDH antibody (ab70699)
This product has been referenced in:
Charron G et al. Prenylome profiling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform. Proc Natl Acad Sci U S A110:11085-90 (2013).
Read more (PubMed: 23776219) »
Yount JS et al. S-Palmitoylation and Ubiquitination Differentially Regulate Interferon-induced Transmembrane Protein 3 (IFITM3)-mediated Resistance to Influenza Virus. J Biol Chem287:19631-41 (2012).
Read more (PubMed: 22511783) »