Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
1/2500 - 1/5000.
1/2500. Detects a band of approximately 40 kDa (predicted molecular weight: 37 kDa).
Some customers have experienced that milk significantly decreases the signal in WB compared to BSA. In-house we use BSA.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration. ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
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.
Predicted band size : 37 kDa Observed band size : 37 kDa
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab9485 overnight at 4°C. Antibody binding was detected using ab175781 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
IHC image of ab9485 staining GAPDH in human pancreas formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. 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 ab9485, 5μg/ml working concentration, 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. No primary antibody was used in the secondary only control (shown on the inset). 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. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody - Loading Control (ab9485)
ab9485 staining GAPDH in NIH3T3 cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes 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 ab9485 at 5μg/ml and ab195889 at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Western blot - Anti-GAPDH antibody - Loading Control (ab9485)
All lanes : Anti-GAPDH antibody - Loading Control (ab9485) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 37 kDa Observed band size : 37 kDa
Exposure time : 10 seconds
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab9485 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody, and visualised using ECL development solution ab133406.
Immunofluorescence - Anti-GAPDH antibody - Loading Control (ab9485)
ab9485 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 ab9485 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-Rabbit secondary (ab150081) 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.
Immunocytochemistry/ Immunofluorescence - GAPDH antibody (ab9485)This image is courtesy of an anonymous Abreview
ab9485 staining GAPDH (green) in Rat bone marrow cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.1% Triton X 100 and blocked with 5% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/250 in PBS) for 12 hours at 4°C. An Alexa Fluor®488-conjugated goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody. Nuclei were stained with DAPI.
Western blot image using the Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-20%) (ab119220) with the Prism Ultra Protein Ladder (ab116028) 5µl used. We recommend using our ECL substrate kit (ab65623) . 20ug of Lysate per lane and detection using ab9485 diluted to 1ug/ml. Lane 1: Hela cell lysate Lane 2: Jurkat cell lysate Lane 3: A431 cell lysate Lane 4: HEK293 cell lysate Lane 5: HepG2 cell lysate.
ICC/IF image of ab9485 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab9485, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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).
Western blot - GAPDH antibody (ab9485)This image is a courtesy of Anonymous Abreview
All lanes : Anti-GAPDH antibody - Loading Control (ab9485) at 1/2500 dilution
Lane 1 : Lysate prepared from human Huh-7 cells at 2 µg Lane 2 : Lysate prepared from human Huh-7 cells at 20 µg
Secondary HRP-conjugated sheep polyclonal to rabbit IgG at 1/20000 dilution
Western blot - GAPDH antibody (ab9485)This picture was generously supplied by Dr Allison Robb of the MRC-LMB, Cambridge, UK.
Predicted band size : 37 kDa
This picture was generously supplied by Dr Allison Robb of the MRC-LMB, Cambridge, UK.
This GAPDH antibody works well with an S. cerevisiae lysate in Western blotting.
A band can be seen down to a dilution of 1/5000.
A: 1:1000, 25 ug lysate B: 1:1000, 50 ug lysate C: 1:2000, 25 ug lysate D: 1:2000, 50 ug lysate E: 1:5000, 25 ug lysate F: 1:5000, 50 ug lysate
References for Anti-GAPDH antibody - Loading Control (ab9485)
This product has been referenced in:
Byun J et al. CR6-interacting factor 1 is a key regulator in Aß-induced mitochondrial disruption and pathogenesis of Alzheimer's disease. Cell Death Differ22:959-73 (2015).
Read more (PubMed: 25361083) »
Asparuhova MB et al. Mechanism of irradiation-induced mammary cancer metastasis: A role for SAP-dependent Mkl1 signaling. Mol Oncol9:1510-27 (2015).
Read more (PubMed: 25999144) »