Overview

  • Product name
    Anti-GAPDH antibody [EPR6256]
    See all GAPDH primary antibodies
  • Description
    Rabbit monoclonal [EPR6256] to GAPDH
  • Tested applications
    Suitable for: WB, IP, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Human, African green monkey
    Does not react with: Mouse
  • Immunogen

    Synthetic peptide corresponding to a region within Human GAPDH (Uniprot: P04406)

  • Positive control
    • WB: 293T, HeLa, HepG2, HUVEC, MCF7 or SH SY5Y cell lysates IF/ICC: MCF7 cells IHC: Hu pancreas
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab128915 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
WB 1/10000 - 1/50000. Detects a band of approximately 35 kDa (predicted molecular weight: 36 kDa).
IP 1/10 - 1/100.
Flow Cyt 1/100 - 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/250 - 1/500.

2.0 µg/ml

IHC-P 1/250.

Target

  • Function
    Has 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.
  • Pathway
    Carbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.
  • Sequence similarities
    Belongs to the glyceraldehyde-3-phosphate dehydrogenase family.
  • Post-translational
    modifications
    S-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus.
    ISGylated.
  • Cellular localization
    Cytoplasm > 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
  • Database links
  • Alternative names
    • 38 kDa BFA-dependent ADP-ribosylation substrate antibody
    • aging associated gene 9 protein antibody
    • Aging-associated gene 9 protein antibody
    • BARS-38 antibody
    • cb609 antibody
    • EC 1.2.1.12 antibody
    • Epididymis secretory sperm binding protein Li 162eP antibody
    • G3P_HUMAN antibody
    • G3PD antibody
    • G3PDH antibody
    • GAPD antibody
    • GAPDH antibody
    • Glyceraldehyde 3 phosphate dehydrogenase antibody
    • Glyceraldehyde-3-phosphate dehydrogenase antibody
    • HEL-S-162eP antibody
    • KNC-NDS6 antibody
    • MGC102544 antibody
    • MGC102546 antibody
    • MGC103190 antibody
    • MGC103191 antibody
    • MGC105239 antibody
    • MGC127711 antibody
    • MGC88685 antibody
    • OCAS, p38 component antibody
    • OCT1 coactivator in S phase, 38-KD component antibody
    • peptidyl cysteine S nitrosylase GAPDH antibody
    • Peptidyl-cysteine S-nitrosylase GAPDH antibody
    • wu:fb33a10 antibody
    see all

Images

  • All lanes : Anti-GAPDH antibody [EPR6256] (ab128915) at 1/10000 dilution

    Lane 1 : 293T cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HepG2 cell lysate
    Lane 4 : HUVEC cell lysate
    Lane 5 : MCF7 cell lysate
    Lane 6 : SH SY5Y cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

    Predicted band size : 36 kDa
    Observed band size : 35 kDa (why is the actual band size different from the predicted?)

    Secondary antibody - anti-rabbit HRP (ab6721)

  • IHC image of ab128915 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 ab128915, 1:250 dilution, 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

  • ab128915 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 ab128915 at 2μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an goat anti-rabbit AlexaFluor®488 (ab150081) at 2 μg/ml (shown in green) and goat anti-mouse AlexaFluor®594 (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.

  • ab128915 staining GAPDH in human HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody. Counter stained with DAPI.

    See Abreview

  • ab128915, at 1/250, staining GAPDH in MCF7 cells by immunofluorescence.
  • Overlay histogram showing HeLa cells stained with ab128915 (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 (ab128915, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was goat anti-rabbit Alexa Fluor® 488 (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

References

This product has been referenced in:
  • Cheng MH  et al. B1, a novel HDAC inhibitor, induces apoptosis through the regulation of STAT3 and NF-?B. Int J Mol Med 39:1137-1148 (2017). Read more (PubMed: 28393178) »
  • Bewicke-Copley F  et al. Extracellular vesicles released following heat stress induce bystander effect in unstressed populations. J Extracell Vesicles 6:1340746 (2017). Read more (PubMed: 28717426) »

See all 19 Publications for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Cell lysate - whole cell (hESC, Cancer-various)
Gel Running Conditions
Reduced Denaturing (10% gel)
Loading amount
100000 cells
Specification
hESC, Cancer-various
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C
Username

Abcam user community

Verified customer

Submitted Aug 18 2015

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Non-reduced Denaturing
Sample
Human Cell lysate - whole cell (lung)
Specification
lung
Treatment
1mM Propanal for 48h
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Ms. Junghee Lim

Verified customer

Submitted Jan 13 2015

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (4-15%)
Sample
Human Cell lysate - whole cell (non small cell lung cancer)
Specification
non small cell lung cancer
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Dr. Satyendra Tripathi

Verified customer

Submitted Sep 25 2014

Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Non-reduced Denaturing (4-12% Nupage gels)
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Sep 18 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
African Green Monkey Cell lysate - whole cell (COS-1 cells)
Specification
COS-1 cells
Treatment
TGF-beta, 24 hours, 50 pM or untreated
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted May 07 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Cell lysate - whole cell (RAMOS cells)
Specification
RAMOS cells
Treatment
TGF-beta, 24 hours, 50 pM or untreated
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted May 07 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (NMuMG cells)
Specification
NMuMG cells
Treatment
TGF-beta, 24 hours, 50 pM or untreated
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted May 07 2014

Application
ELISA
Sample
Human Recombinant protein (HEK293)
Specification
HEK293
Type
Sandwich (Detection)
Blocking step
SuperBlock PBS Pierce #37580 as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10µg/mL · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Mar 14 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (8)
Sample
Mouse Cell lysate - whole cell (mouse embryonic fibroblasts (MEFs))
Specification
mouse embryonic fibroblasts (MEFs)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Feb 21 2014

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Permeabilization
Yes - 0.5% Triton X100 in PBS
Fixative
Paraformaldehyde
Username

Dr. Kirk Mcmanus

Verified customer

Submitted Jan 22 2014

1-10 of 11 Abreviews or Q&A

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