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

Overview

  • Product name
    Anti-GAPDH antibody [mAbcam 9484] - Loading Control
    See all GAPDH primary antibodies
  • Description
    Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control
  • Host species
    Mouse
  • Tested applications
    Suitable for: 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 (purified) corresponding to Human GAPDH.

  • General notes

    For Western blotting, do not use milk for blocking. Our labs have extensively tested the blocking conditions for this antibody and recommend using 5% BSA for 1 hour. The comparison data are shown in the images section. 

    This antibody clone [mAbcam 9484] is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

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
WB Use a concentration of 0.1 - 1 µg/ml. Predicted molecular weight: 36 kDa.

Do not block with milk. Block with 5% BSA for 1 hour. Our labs have thoroughly investigated the blocking conditions for this antibody. We found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see images).

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.

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody

Target

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

    Performed under reducing conditions.

    Predicted band size: 36 kDa
    Observed band size: 40 kDa (why is the actual band size different from the predicted?)


    Exposure time: 10 seconds


    The membrane was blocked in 5% BSA in TBST for 1 hour, then incubated for 1 hour in primary antibody diluted in TBST.

  • 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
    All lanes : 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: 36 kDa
    Observed band size: 41 kDa (why is the actual band size different from the predicted?)


    Exposure time: 1 minute

    See 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
    All lanes : Rat anti-rabbit IgG (H+L) IgG polyclonal at 1/5000 dilution

    Developed using the ECL technique.

    Predicted band size: 36 kDa
    Observed band size: 41 kDa (why is the actual band size different from the predicted?)


    Exposure time: 1 minute

    See Abreview

  • 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)

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

    Lysates/proteins at 20 µg per lane.

    Secondary
    Lanes 1-5 : Goat anti-Mouse (HRP conjugated) at 1/5000 dilution
    Lanes 6-10 : Goat anti-Mouse (HRP conjugated) at 1/5000 dilution

    Predicted band size: 36 kDa
    Observed band size: 40 kDa (why is the actual band size different from the predicted?)



    The membrane 1-5 was blocked in 5% milk (1 hour). The membrane 6-10 was blocked in 5% BSA (1 hour). Milk is not a suitable blocking agent and significantly decreases the signal on the membrane.

  • 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: 36 kDa


    Exposure time: 30 seconds
  • 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.
  • 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

  • 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

This product has been referenced in:
  • Krysiak J  et al. Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes. Nat Commun 9:262 (2018). Read more (PubMed: 29343782) »
  • Subkhangulova A  et al. SORCS1 and SORCS3 control energy balance and orexigenic peptide production. EMBO Rep 19:N/A (2018). WB . Read more (PubMed: 29440124) »

See all 369 Publications for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Cell lysate - whole cell (liver, HepG2)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
liver, HepG2
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 Dec 04 2017

Application
Western blot
Sample
Rat Tissue lysate - whole (Retina)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
20 µg
Specification
Retina
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Aug 01 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Zebrafish Tissue lysate - whole (Liver, RPE, retina)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
20 µg
Specification
Liver, RPE, retina
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 24 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK cells)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
20 µg
Specification
HEK cells
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 17 2017

Application
Western blot
Sample
Mouse Tissue lysate - whole (Liver)
Gel Running Conditions
Reduced Denaturing (10% gel)
Loading amount
20 µg
Specification
Liver
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Dec 14 2016

Application
Western blot
Sample
Rat Tissue lysate - whole (muscle)
Gel Running Conditions
Reduced Denaturing (9)
Loading amount
50 µg
Specification
muscle
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Mr. Baptiste Jude

Verified customer

Submitted Oct 07 2016

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

1-10 of 67 Abreviews or Q&A

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