Recombinant
RabMAb

Recombinant Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915)

Rabbit recombinant monoclonal GAPDH antibody [EPR6256]. Validated in WB, IP, IHC, Flow Cyt, ICC/IF and tested in Human, African green monkey. Cited in 53 publication(s).

Overview

  • Product name
    Anti-GAPDH antibody [EPR6256] - Loading Control
    See all GAPDH primary antibodies
  • Description
    Rabbit monoclonal [EPR6256] to GAPDH - Loading Control
  • Host species
    Rabbit
  • Specificity

    The African green monkey recommendation is based on the WB results. We do not guarantee IHC-P for African green monkey.

  • Tested applications
    Suitable for: WB, IP, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Human, African green monkey
  • Immunogen

    Synthetic peptide within Human GAPDH aa 250 to the C-terminus. The exact sequence is proprietary.
    Database link: P04406

  • Positive control
    • WB: 293T, HeLa, HepG2, HUVEC, MCF7 and SH-SY5Y cell lysates. ICC/IF: HeLa and MCF7 cells. IHC-P: Human pancreas tissue, Human bladder carcinoma. Flow Cyt: HeLa cells. IP: HeLa cell lysate.
  • General notes

    Mouse: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    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 1/20.

For unpurified use at 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 1/2000.

See IHC antigen retrieval protocols.

For unpurified use at 1/250.

The African green monkey recommendation is based on the WB results. We do not guarantee IHC-P for African green monkey.

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

  • Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/20000 dilution (Purified) + COS-1 (African green monkey  kidney fibroblast-like) whole cell lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

  • Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/20000 dilution (Purified) + HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

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

    Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
    Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
    Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate
    Lane 4 : HUVEC (human umbilical vein endothelial cell line) cell lysate
    Lane 5 : MCF7 (human breast adenocarcinoma cell line) cell lysate
    Lane 6 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : 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)

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling GAPDH with purified ab128915 at 1/2000 dilution (0.06 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GAPDH with purified ab128915 at 1/250 dilution (0.4 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 (2.5 µg/ml) dilution. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GAPDH with purified ab128915 at 1/20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • ab128915 (purified) at 1/20 dilution (0.5ug) immunoprecipitating GAPDH in HeLa whole cell lysates.
    Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates 10ug
    Lane 2 (+): ab128915 & HeLa whole cell lysates
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab128915 in HeLa whole cell lysates
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • IHC image of ab128915 (unpurified) 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 (unpurified) staining GAPDH in HeLa (human epithelial cell line from cervix adenocarcinoma) 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.

  • Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab128915 (unpurified) (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.

  • ab128915 (unpurified) staining GAPDH in human HeLa (human epithelial cell line from cervix adenocarcinoma) 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 (unpurified), at 1/250, staining GAPDH in MCF7 (human breast adenocarcinoma cell line) cells by immunofluorescence.

References

This product has been referenced in:
  • Gao Q  et al. Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer. J Exp Med 216:688-703 (2019). Read more (PubMed: 30710055) »
  • Guo L  et al. MicroRNA-663b targets GAB2 to restrict cell proliferation and invasion in hepatocellular carcinoma. Mol Med Rep 19:2913-2920 (2019). Read more (PubMed: 30720118) »
See all 68 Publications for this product

Customer reviews and Q&As

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1-10 of 11 Abreviews

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

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

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

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

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

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

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

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

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

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

Dr. Kirk Mcmanus

Verified customer

Submitted Jan 22 2014

1-10 of 11 Abreviews

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