Anti-GAPDH antibody - Loading Control (ab9485)

Overview

  • Product nameAnti-GAPDH antibody - Loading Control
    See all GAPDH primary antibodies
  • Description
    Rabbit polyclonal to GAPDH - Loading Control
  • Tested applicationsIP, 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 from human erythrocytes.

  • Positive control
    • WB: HeLa, A431, A549, NIH3T3, PC12 whole cell lysate ICC: U2OS cells ICC/IF: HeLa cells

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
IF Use a concentration of 5 µg/ml.
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.

IHC-Fr 1/250.
ICC/IF Use a concentration of 5 µg/ml.
Flow Cyt Use at an assay dependent concentration.

Target

  • 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
    modifications
    S-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
  • Database links
  • Alternative names
    • Aging associated gene 9 protein antibody
    • G3P_HUMAN antibody
    • G3PD antibody
    • G3PD antibody
    • G3PDH antibody
    • GAPD antibody
    • GAPDH antibody
    • GAPDH antibody
    • Glyceraldehyde 3 phosphate dehydrogenase antibody
    • Glyceraldehyde 3 phosphate dehydrogenase liver antibody
    • Glyceraldehyde 3 phosphate dehydrogenase muscle antibody
    • Glyceraldehyde-3-phosphate dehydrogenase antibody
    • MGC88685 antibody
    • OCAS p38 component antibody
    • OCT1 coactivator in S phase 38 KD Component antibody
    • Peptidyl-cysteine S-nitrosylase GAPDH antibody
    see all

Anti-GAPDH antibody - Loading Control images

  • 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.

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

  • 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 antibody conjugated to HRP, and visualised using ECL development solution ab133406.

  • 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.

    See Abreview



  • Predicted band size : 37 kDa

    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).

  • 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

    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

    This image is a courtesy of Anonymous Abreview

    See Abreview



  • 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:
  • Kim J  et al. Memory recall and modifications by activating neurons with elevated CREB. Nat Neurosci 17:65-72 (2014). Read more (PubMed: 24212670) »
  • Lojewski X  et al. Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects of TPP1 and CLN3 mutations on the endocytic pathway. Hum Mol Genet 23:2005-22 (2014). WB ; Human . Read more (PubMed: 24271013) »

See all 176 Publications for this product

Product Wall

Thank you for contacting us and for providing these additional details. Do you fix and permeablize your nuclear extracts or are the nuclear envelopes removed prior to staining? While I would recommend using a commercial fix and permeablization solut...

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Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (primary lung cancer lines)
Specification primary lung cancer lines
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 25 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (lung cancer lines)
Specification lung cancer lines
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 25 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (hepatocytes)
Specification hepatocytes
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Jul 04 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 50 µg
Gel Running Conditions Non-reduced Non-Denaturing (Native) (10 % gel)
Sample Rat Tissue lysate - whole (rat baldder)
Specification rat baldder
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Dr. EUN-YOUNG PARK

Verified customer

Submitted Jul 18 2013

The chicken antibody may be a suitable capture antibody but it has not been tested with ab8245. The difference with ab9485, the rabbit polyclonal, is that ab83956, the chicken antibody, is raised against a peptide from the carboxy terminus of GAPDH. Th...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (B-lymphocytes)
Loading amount 20 µg
Specification B-lymphocytes
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Dr. Elena Kashuba

Verified customer

Submitted Apr 03 2013

I would be very happy to provide an alternative product against GAPDH in E.coli. We do carry one anti-GAPDH which is tested and guaranteed in E.coli. This product is ab85760. This product is conjugated to HRP so you will not need to use a secondary ant...

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I am sorry to hear that you have been experiencing troubles when using this product in E.coli. As we have not yet tested in this species we cannot be sure how effective the antibody will be and therefore cannot guarantee that you will be able to achiev...

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Thank you for contacting us. Yes, this product is suitable for your purposes. Please let me know if you have any further questions.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"