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

Overview

  • Product nameAnti-GAPDH [mAbcam 9484] antibody - Loading ControlSee all GAPDH primary antibodies ...
  • Description
    Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control
  • Tested applicationsICC/IF, WB, IHC-P, Flow Cyt more 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 Notes
ICC/IF Use a concentration of 5 µg/ml.
WB Use a concentration of 0.5 - 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 [mAbcam 9484] antibody - Loading Control images

  • All lanes : Anti-GAPDH [mAbcam 9484] antibody - 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

    The membrane was blocked in 5%BSA in TBST for one hour, then incubated for one hour in primary antibody diluted in TBST.
  • Anti-GAPDH [mAbcam 9484] antibody - 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 [mAbcam 9484] antibody - Loading Control (ab9484) at 1/1000 dilution (Blocked in 5% MILK)
    Lanes 6 - 10 : Anti-GAPDH [mAbcam 9484] antibody - 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
  • 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, DyLight® 488 goat anti-mouse 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 DyLight® 488 goat anti-mouse 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 [mAbcam 9484] antibody - 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) »
  • Dzinic SH  et al. Identification of an intrinsic determinant critical for maspin subcellular localization and function. PLoS One 8:e74502 (2013). Human . Read more (PubMed: 24278104) »

See all 100 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (15)
Sample Human Tissue lysate - whole (Brain)
Specification Brain
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 31 2014

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Non-Denaturing (Native) (12)
Sample Mouse Cell lysate - whole cell (Gastrocnemius)
Specification Gastrocnemius
Blocking step Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Mr. FRANCESCO ELIA MARINO

Verified customer

Submitted Mar 12 2014

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - 0.1% Triton in PBS
Sample Human Cell (HL-1 (Cardiomyocyte line))
Specification HL-1 (Cardiomyocyte line)
Gating Strategy isotype control (black line in histogram)
Preparation Cell harvesting/tissue preparation method: accutase and accumax dissociation
Sample buffer: PBS 7.4 + 10% FBS for washing
Username

Abcam user community

Verified customer

Submitted Mar 10 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (12% gel)
Sample Mouse Tissue lysate - whole (Skeletal muscle)
Specification Skeletal muscle
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jan 21 2014

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

Dr. G.K Sakellariou

Verified customer

Submitted May 14 2013

Thank you for contacting us.

We have published safety datasheets for these products on the website. These will be available soon. Please download these from our website.

I hope this information is helpful to you. Please do not hesitat...

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Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse ES Cells)
Loading amount 50 µg
Specification Mouse ES Cells
Gel Running Conditions Reduced Denaturing (12)
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Mr. Utkarsh Kapoor

Verified customer

Submitted Dec 17 2012

Application Western blot
Sample Mouse Tissue lysate - whole (Skeletal muscle (GTN))
Loading amount 50 µg
Specification Skeletal muscle (GTN)
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Dr. G.K Sakellariou

Verified customer

Submitted Sep 28 2012

Thank you for contacting us. We have not tested this product in Syrian hamster, however given the high homology with the human sequence it is likely to react.


I hope this information is helpful to you. Please do not hesitate to contact us...

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Thank you for confirming that.

As discussed over the phone, I have arranged for a replacement vial to be sent to you. This is on the order number xxxxx(Purchase order number xxxxxx). This has been dispatched and should be with you tomorrow. <...

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