Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)
- Datasheet
- References (464)
- Protocols
Overview
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Product nameAnti-GAPDH antibody [mAbcam 9484] - Loading Control
See all GAPDH primary antibodies -
DescriptionMouse monoclonal [mAbcam 9484] to GAPDH - Loading Control
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Host speciesMouse
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Specificity
In western blot, this product typically gives a lower signal in rat lysates compared to human and mouse lysates.
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Tested applicationsSuitable for: WB, IHC-P, Flow Cytmore details
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Species reactivityReacts with: Mouse, Rat, Rabbit, Chicken, Cow, Dog, Human, Pig, Xenopus laevis, Cynomolgus monkey, Chinese hamster
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Immunogen
Full length native protein (purified) corresponding to Human GAPDH.
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General notes
For Western blotting, do not use milk for blocking. Our labs have extensively tested the blocking conditions for this antibody and recommend using 5% BSA for 1 hour. The comparison data is shown in the images section.
This antibody clone [mAbcam 9484] is manufactured by Abcam.
If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.
Properties
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FormLiquid
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
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Storage bufferpH: 7.4
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine -
Concentration information loading... -
PurityIgG fraction
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ClonalityMonoclonal
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Clone numbermAbcam 9484
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MyelomaSp2/0-Ag14
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IsotypeIgG2b
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Light chain typekappa
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Research areas
Associated products
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Alternative Versions
- Anti-GAPDH antibody [mAbcam 9484] - BSA and Azide free (ab174646)
- Anti-GAPDH antibody [mAbcam 9484] - Loading Control (Alexa Fluor® 680) (ab184095)
- Anti-GAPDH antibody [mAbcam 9484] - Loading Control (Alexa Fluor® 790) (ab184578)
- Anti-GAPDH antibody [mAbcam 9484] (Alexa Fluor® 647) (ab196697)
- Anti-GAPDH antibody [mAbcam 9484] - Loading Control (HRP) (ab9482)
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Compatible Secondaries
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Conjugation kits
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Immunohistochemistry kits
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Isotype control
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Recombinant Protein
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Related Products
Applications
Our Abpromise guarantee covers the use of ab9484 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
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| WB | Use a concentration of 0.1 - 1 µg/ml. Predicted molecular weight: 36 kDa. Do not block with milk. Block with 5% BSA for 1 hour. Our labs have thoroughly investigated the blocking conditions for this antibody. We found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see images). |
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| IHC-P | Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. | |
| Flow Cyt | Use 1µg for 106 cells. ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody. We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody |
Target
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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.
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PathwayCarbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.
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Sequence similaritiesBelongs to the glyceraldehyde-3-phosphate dehydrogenase family.
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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.
- Information by UniProt
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Database links
- Entrez Gene: 374193 Chicken
- Entrez Gene: 281181 Cow
- Entrez Gene: 403755 Dog
- Entrez Gene: 2597 Human
- Entrez Gene: 100042025 Mouse
- Entrez Gene: 14433 Mouse
- Entrez Gene: 396823 Pig
- Entrez Gene: 100009074 Rabbit
see all -
Alternative names
- 38 kDa BFA-dependent ADP-ribosylation substrate antibody
- aging associated gene 9 protein antibody
- Aging-associated gene 9 protein antibody
see all
Images
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![Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)](https://www.abcam.com/ps/products/9/ab9484/Images/ab9484-344444-DS4ab9484AP41529711m.jpg)
Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)All lanes : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1 µg/ml
Lane 1 : HeLa Whole Cell Lysate
Lane 2 : NIH 3T3 Whole Cell Lysate
Lane 3 : PC12 Whole Cell Lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 36 kDa
Exposure time: 1 minuteThis 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 ab9484 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406
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![Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)](https://www.abcam.com/ps/products/9/ab9484/Images/ab9484-17140-anti-gapdh-antibody-mabcam-9484-loading-control-western-blot.jpg)
Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)All lanes : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/5000 dilution
Lane 1 : Hela whole cell (Human)
Lane 2 : 3T3 cell (Mouse)
Lane 3 : Rat brain
Lane 4 : Xenopus embryo
Lane 5 : Chicken Liver
Lane 6 : EBTr cell (Cow)
Lane 7 : CHO cell (Chinese hamster)
Lane 8 : Pig liver
Secondary
All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?
Exposure time: 10 secondsThe membrane was blocked in 5% BSA in TBST for 1 hour, then incubated for 1 hour in primary antibody diluted in TBST.
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![Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)](https://www.abcam.com/ps/products/9/ab9484/Images/ab9484-17138-anti-gapdh-antibody-mabcam-9484-loading-control-western-blot.jpg)
Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)Lanes 1-5 : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/1000 dilution (Blocked in 5% milk)
Lanes 6-10 : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/1000 dilution (Blocked in 5% BSA)
Lanes 1 & 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lanes 2 & 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lanes 3 & 8 :A431 whole cell lysate (ab7909)
Lanes 4 & 9 :Jurkat whole cell lysate (ab7899)
Lanes 5 & 10 :HEK293 whole cell lysate (ab7902)
Lysates/proteins at 20 µg per lane.
Secondary
Lanes 1-5 : Goat anti-Mouse (HRP conjugated) at 1/5000 dilution
Lanes 6-10 : Goat anti-Mouse (HRP conjugated) at 1/5000 dilution
Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?The membrane 1-5 was blocked in 5% milk (1 hour). The membrane 6-10 was blocked in 5% BSA (1 hour). Milk is not a suitable blocking agent and significantly decreases the signal on the membrane.
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![Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)](https://www.abcam.com/ps/products/9/ab9484/Images/ab9484-17139-anti-gapdh-antibody-mabcam-9484-loading-control-western-blot.jpg)
Western blot - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 0.5 µg/ml + HeLa cell lysate
Secondary
Goat Anti-Mouse IgG H&L (HRP) (ab6789) at 1/5000 dilution
Developed using the ECL technique.
Performed under non-reducing conditions.
Predicted band size: 36 kDa
Exposure time: 30 seconds -
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)](https://www.abcam.com/ps/products/9/ab9484/Images/ab9484-17137-anti-gapdh-antibody-mabcam-9484-loading-control-immunohistochemistry.jpg)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)IHC image of GAPDH staining in human liver FFPE section, performed on a 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 ab9484, 5µg/ml, for 8 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. -
![Flow Cytometry - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)](https://www.abcam.com/ps/products/9/ab9484/Images/ab9484-17133-anti-gapdh-antibody-mabcam-9484-loading-control-flow-cytometry.jpg)
Flow Cytometry - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)Overlay histogram showing HeLa cells stained with ab9484 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab9484, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was goat anti-mouse DyLight® 488 (IgG H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Protocols
References
This product has been referenced in:
- Gordon D et al. Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction. Neurobiol Dis 121:148-162 (2019). Read more (PubMed: 30290270) »
- Mulati K et al. VISTA expressed in tumour cells regulates T cell function. Br J Cancer 120:115-127 (2019). Read more (PubMed: 30382166) »
