Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

Overview

Properties

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
ICC/IF Use a concentration of 5 µg/ml.
WB Use a concentration of 0.1 - 1 µg/ml. Detects a band of approximately 36 kDa (predicted molecular weight: 40.2 kDa).

NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have thoroughly investigated the blocking conditions for this antibody following concerning customer feedback on the lack of signal with some of the vials. We found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see images). This change in the characteristics of the antibody is due to a recent update to the production process.

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.

Target

Anti-GAPDH antibody [mAbcam 9484] - Loading Control images

  • 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
    Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 40.2 kDa


    Exposure time : 10 seconds

    Western blot using ab9484 at 1/5000.

    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 ab: Rabbit polyclonal to mouse IgG (ab6728 at 1/5000). Exposure time: 10 seconds.

    The membrane was blocked in 5%BSA in TBST for one hour, then incubated for one hour in primary antibody diluted in TBST.

     

    Secondary antibody - rabbit anti-mouse HRP (ab6728)

     

     

  • Immunohistohemical staining of human ovary cystadenocarcinoma with ab9484 at 1/200. Samples were incubated with the primary antibody for 14 hours at 4ºC in PBS/5% goat serum. A HRP conjugated goat anti-mouse was used as the secondary at a dilution of 1/2000.

    See Abreview

  • All lanes : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/2000 dilution

    Lane 1 : Chinese hamster ovary whole cell lysates
    Lane 2 : Bortezomib treated (100nM for 16 hours) Chinese hamster ovary whole cell lysates

    Secondary
    HRP-conjugated goat anti-mouse polyclonal IgG (H+L) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 40.2 kDa
    Additional bands at : 41 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview.

  • All lanes : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/200 dilution

    Lane 1 : Rat intestinal epithelial (RIE-1) whole cell lysates
    Lane 2 : Bortezomib treated (100nM for 16 hours) rat intestinal epithelial (RIE-1) whole cell lysates

    Lysates/proteins at 100000 cells per lane.

    Secondary
    Rat anti-rabbit IgG (H+L) IgG polyclonal at 1/5000 dilution
    developed using the ECL technique

    Predicted band size : 40.2 kDa
    Additional bands at : 41 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview.

  • 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 : 40.2 kDa


    Exposure time : 30 seconds
  • 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)

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : A431 whole cell lysate (ab7909)
    Lane 4 : Jurkat whole cell lysate (ab7899)
    Lane 5 : HEK293 whole cell lysate (ab7902)
    Lane 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
    Lane 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 8 : A431 whole cell lysate (ab7909)
    Lane 9 : Jurkat whole cell lysate (ab7899)
    Lane 10 : HEK293 whole cell lysate (ab7902)

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat anti-Mouse (HRP conjugated) at 1/5000 dilution

    Predicted band size : 40.2 kDa
    Observed band size : 40.2 kDa
    The membrane 1-5 was blocked in 5% Milk (1 hour). The membrane 6-10 was blocked in 5% BSA (1 hour). Abcam routinely uses 5% BSA to block however following recent customer feedback our labs investigated the effect of 5% milk blocking. We can now confirm that milk is not a suitable blocking agent and significantly decreases the signal on the membrane. We have informed all users of the antibody of this finding.
  • 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.
  • ICC/IF image of ab9484 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9484, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ICC/IF image of ab9484 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9484, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, anti mouse-DyLight® 488 (IgG H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Flow Cytometry analysis of GAPDH using ab9484.
    Human monocytes were fixed in paraformaldehyde and permeabilized. ab9484 was used at a 1/200 dilution. The secondary used was an Alexa-Fluor 488 conjugated chicken anti-rabbit IgG (H+L) polyclonal, used at a 1/500 dilution.

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  • 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 22ºC. The secondary antibody used was goat anti-mouse DyLight® 488 (IgG H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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.

  • ab9484 staining GAPDH in A549 lung cancer cells by Flow Cytometry. Cells were harvested in trypsin, fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100. The sample was incubated with the primary antibody (1/100 in 1x HBSS + 0.02% Triton X-100 + 1.5% FBS) for 3 hours at 25°C. An Alexa Fluor® 488-conjugated goat anti-mouse IgG polyclonal (1/2000) was used as the secondary antibody.
    Gating Strategy: No gating.

    See Abreview

References for Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

This product has been referenced in:
  • García-Bea A  et al. Metabotropic glutamate receptor 3 (mGlu3; mGluR3; GRM3) in schizophrenia: Antibody characterisation and a semi-quantitative western blot study. Schizophr Res N/A:N/A (2016). WB ; Human . Read more (PubMed: 27130562) »
  • Tanco S  et al. C-terminomics screen for natural substrates of cytosolic carboxypeptidase 1 reveals processing of acidic protein C termini. Mol Cell Proteomics 14:177-90 (2015). Read more (PubMed: 25381060) »

See all 144 Publications for this product

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Thank you for contacting us. The recommended dilution of 1:150 is a recommendation which we make to our customers based on our experiments or those of supplier, collaborator or customer input. Although we cannot know exactly why certain products req...

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Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (H1299 Lung cancer cell line)
Permeabilization Yes - 0.1% v/v TritonX-100
Specification H1299 Lung cancer cell line
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative Paraformaldehyde
Username

Dr. Dimitra Kalamida

Verified customer

Submitted Jun 06 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (PERIPHERAL BLOOD MONONUCLEAR CELLS)
Gel Running Conditions Reduced Denaturing (12% SDS PAGE)
Loading amount 30 µg
Specification PERIPHERAL BLOOD MONONUCLEAR CELLS
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Siddharth Mehra

Verified customer

Submitted Jun 02 2016

Application Western blot
Sample Rat Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions Reduced Denaturing (12%)
Loading amount 20 µg
Specification Skeletal muscle
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 17 2016

Application Western blot
Sample Mouse Cell lysate - whole cell (mouse hepatocytes)
Gel Running Conditions Reduced Denaturing
Loading amount 25 µg
Specification mouse 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 Mar 14 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Chinese Hamster Cell lysate - whole cell (Chinese hamster ovary cell)
Gel Running Conditions Reduced Denaturing (4-12% Bis-Tris)
Loading amount 100000 cells
Treatment 100 nM Bortezomib for 16 hours
Specification Chinese hamster ovary cell
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 10 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Rat Cell lysate - whole cell (rat intestinal epithelial cells (RIE-1))
Gel Running Conditions Reduced Denaturing (4-12% Bis-Tris)
Loading amount 100000 cells
Treatment 100 nM Bortezomib for 16 hours
Specification rat intestinal epithelial cells (RIE-1)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 10 2016

Application Western blot
Sample Human Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions Reduced Denaturing (12%)
Loading amount 15 µg
Specification Skeletal muscle
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 02 2016

Application Flow Cytometry
Sample Mouse Cell (Mouse Lewis Lung Carcinoma cells)
Permeabilization Yes - 0.2% Triton X-100
Gating Strategy No gating
Specification Mouse Lewis Lung Carcinoma cells
Preparation Cell harvesting/tissue preparation method: trypsin
Sample buffer: Hanks’ Balanced Salt solution (HBSS)
Fixation Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 25 2016

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