製品の概要

  • 製品名
    Anti-GAPDH antibody [mAbcam 9484] - Loading Control
    GAPDH 一次抗体 製品一覧
  • 製品の詳細
    Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control
  • 由来種
    Mouse
  • アプリケーション
    適用あり: WB, IHC-P, Flow Cytmore details
  • 種交差性
    交差種: Mouse, Rat, Rabbit, Chicken, Cow, Dog, Human, Pig, Xenopus laevis, Cynomolgus monkey, Chinese hamster
  • 免疫原

    Full length native protein (purified) corresponding to Human GAPDH.

  • 特記事項

    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.

    Abcam recommended secondaries - Goat Anti-Mouse HRP (ab205719) and Goat Anti-Mouse Alexa Fluor® 488 (ab150113).

    See other anti-mouse secondary antibodies that can be used with this antibody.

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
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

ターゲット情報

画像

  • 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.

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

参考文献

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 430 Publications for this product

レビューと Q&A

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1-10 of 60 Abreviews

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

Abcam user community

Verified customer

投稿 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

Abcam user community

Verified customer

投稿 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

Abcam user community

Verified customer

投稿 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

Abcam user community

Verified customer

投稿 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

Abcam user community

Verified customer

投稿 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

Mr. Baptiste Jude

Verified customer

投稿 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

Dr. Dimitra Kalamida

Verified customer

投稿 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

Mr. Siddharth Mehra

Verified customer

投稿 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

Abcam user community

Verified customer

投稿 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

Abcam user community

Verified customer

投稿 Mar 14 2016

1-10 of 60 Abreviews

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

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