Overview

  • Product name
    Anti-GAPDH antibody - Loading Control
    See all GAPDH primary antibodies
  • Description
    Rabbit polyclonal to GAPDH - Loading Control
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, IP, ELISA, WB, IHC-Fr, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Dog, Human, Saccharomyces cerevisiae, Xenopus laevis, Schizosaccharomyces pombe, African green monkey
  • Immunogen

    Full length native protein (purified) corresponding to Human GAPDH.

  • Positive control
    • WB: HeLa, A431, A549, NIH3T3, PC12 whole cell lysate ICC: U2OS cells ICC/IF: HeLa cells, NIH3T3 cells IHC/P: Hu Pancreas (FFPE)
  • General notes

      

Properties

Applications

Our Abpromise guarantee covers the use of ab9485 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
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IP 1/250.
ELISA 1/2500 - 1/5000.
WB 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.

We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) secondary antibody.

IHC-Fr 1/250.
ICC/IF Use a concentration of 5 µg/ml.

We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody.

Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

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 - Loading Control (ab9485) at 1/2500 dilution

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) secondary antibody at 1/10000 dilution

    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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) secondary antibody at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) secondary antibody 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.

  • 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

  • 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody 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 image using 4-20% Optiblot gel 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.

  • 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
    All lanes : HRP-conjugated sheep polyclonal to rabbit IgG at 1/20000 dilution

    Performed under reducing conditions.

    Predicted band size: 37 kDa
    Observed band size: 40 kDa
    why is the actual band size different from the predicted?


    Exposure time: 5 minutes

    See Abreview

  • 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
    All lanes : 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 Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody, and visualised using ECL development solution ab133406.

References

This product has been referenced in:
  • Aubi O  et al. Early Stage Discovery and Validation of Pharmacological Chaperones for the Correction of Protein Misfolding Diseases. Methods Mol Biol 1873:279-292 (2019). Read more (PubMed: 30341617) »
  • Negoita F  et al. PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes. J Cell Biochem 120:343-356 (2019). Read more (PubMed: 30171718) »
See all 1067 Publications for this product

Customer reviews and Q&As

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1-10 of 83 Abreviews

Application
Western blot
Sample
Human Cell lysate - whole cell (primary cardiomyocytes)
Gel Running Conditions
Reduced Denaturing (4-12 %)
Loading amount
20 µg
Treatment
LPS 1 µg/ml
Specification
primary cardiomyocytes
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Miss. Barbara Thaler

Verified customer

Submitted Jul 04 2019

Application
Western blot
Sample
Mouse Cell lysate - whole cell (HL-1 cardiomyocytes)
Gel Running Conditions
Reduced Denaturing (4-12 %)
Loading amount
40 µg
Treatment
LPS 1 µg/ml
Specification
HL-1 cardiomyocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Miss. Barbara Thaler

Verified customer

Submitted Jul 03 2019

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Primary murine microglia)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Treatment
1µM LPA for different time points
Specification
Primary murine microglia
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Dr. Lisha Joshi

Verified customer

Submitted Jun 12 2019

Application
Western blot
Sample
Human Cell lysate - whole cell (PNT2 prostate tissue)
Gel Running Conditions
Reduced Denaturing (8)
Loading amount
20 µg
Specification
PNT2 prostate tissue
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jan 08 2019

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mouse brain primary culture)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
400000 cells
Specification
Mouse brain primary culture
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 28°C

Abcam user community

Verified customer

Submitted Dec 31 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (Human A431 cells)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
20 µg
Specification
Human A431 cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 22 2018

Application
Western blot
Sample
Mouse Tissue lysate - whole (Liver)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (10%)
Loading amount
35 µg
Treatment
5% ethanol for 10 days
Specification
Liver
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Oct 23 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (H1944 lung adenocarcinoma)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
18 µg
Specification
H1944 lung adenocarcinoma
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 20°C

Dr. Wei-Ting Lu

Verified customer

Submitted Aug 20 2018

Application
Western blot
Sample
Cryptococcus neoformans Cell lysate - other (Subcellular fractionations)
Gel Running Conditions
Reduced Denaturing (Criterion TGX 4-20%)
Loading amount
40 µg
Specification
Subcellular fractionations
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jan 30 2018

Application
Western blot
Sample
Mouse Cell lysate - whole cell (BV2)
Gel Running Conditions
Non-reduced Denaturing (10)
Loading amount
100 µg
Treatment
10 nM for 24hrs
Specification
BV2
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Dr. jianhai long

Verified customer

Submitted Jan 12 2018

1-10 of 83 Abreviews

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