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

Overview

  • Product nameAnti-GAPDH antibody [mAbcam 9484] - Loading Control
    See all GAPDH primary antibodies
  • Description
    Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control
  • Tested applicationsICC/IF, WB, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Chicken, Cow, Dog, Human, Pig, Xenopus laevis, Cynomolgus Monkey, Chinese Hamster
  • Immunogen

    Full length native protein from human erythrocytes.

  • General notes

    We can conjugate this antibody to FITC for you (please see ab150196 for details).

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.

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)

     

     

  • 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 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)
    Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
    Lane 5 : HEK293 (Human embryonic kidney cell line) 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 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)
    Lane 9 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
    Lane 10 : HEK293 (Human embryonic kidney cell line) 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.

    See Abreview

  • 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.

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

This product has been referenced in:
  • Davoudi M  et al. Complex I function and supercomplex formation are preserved in liver mitochondria despite progressive complex III deficiency. PLoS One 9:e86767 (2014). WB ; Mouse . Read more (PubMed: 24466228) »
  • Radin JN  et al. Role of connexin 43 in Helicobacter pylori VacA-induced cell death. Infect Immun 82:423-32 (2014). Human . Read more (PubMed: 24191302) »

See all 128 Publications for this product

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Thank you for providing the Western blot images.
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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 Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Chinese Hamster Cell lysate - whole cell (CHO cell)
Specification CHO cell
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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Submitted Nov 04 2014

Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample Mouse Tissue sections (mouse heart)
Specification mouse heart
Permeabilization Yes - 1% TRITON-X-100
Fixative Paraformaldehyde
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Submitted Sep 30 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: 0.1 M citration buffer
Sample Mouse Tissue sections (mouse heart)
Specification mouse heart
Permeabilization Yes - 1% TRITON-X-100
Fixative Formaldehyde
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Submitted Sep 24 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Rat Cell lysate - whole cell (h9c2)
Specification h9c2
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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Submitted Sep 24 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (HEK293)
Specification HEK293
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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Submitted Sep 24 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (MEF)
Specification MEF
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

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Submitted Sep 24 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12% gel)
Sample Rat Tissue lysate - other (Cytosolic fractions from skeletal muscles)
Specification Cytosolic fractions from skeletal muscles
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Jun 02 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Odyssey Blocking Bufer as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C
Sample Human Cell (COV434 cell line)
Specification COV434 cell line
Permeabilization Yes - Ethanol
Fixative Paraformaldehyde
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Mr. FRANCESCO ELIA MARINO

Verified customer

Submitted May 30 2014

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