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.

    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.

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.

  • 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.

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 430 Publications for this product

Customer reviews and Q&As

1-7 of 7 Q&A

Answer

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with one of our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints. I appreciate the time taken to submit further information to us, and after reading the answers provided I would like to ask some additional questions.

What type of sample are you using (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) and how is the sample prepared (Buffer/Protease inhibitors/Heating sample etc.)?

How much protein is loaded in each well?

You mention BSA blocking, have you tried other blocking conditions or periods?

How much primary antibody did you use? How long and at what temperature was this incubated?

What dilution and incubation did you use your secondary antibody at?

What detection method do you use (ECL, ECLPlus etc.)?

Have you run a no primary antibody control?

Do you obtain the same results every time (e.g. are the background bands always in the same place)?

We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.


Please let me know if you have any questions. I look forward to hearing from you.

Read More

Answer

Thank you for your inquiry. We do not assign strict expiration dates for our products, however we follow general guidelines: Antibodies stored at 4C may be viable up to 1 year, those stored at -20C may be viable for up to 5 years, and those stored at -80C may be viable for up to 10 years. We guarantee our antibodies for tested species and applications for 12 months from purchase under the Abpromise guarantee.

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Answer

You are right in stating that customers have reported successful experiments using milk as a blocking agent. We have however changed our production methods for this monoclonal antibody and this appears to have altered the affinity of the antibody somewhat. We now strongly recommend using BSA instead of milk, as in our hands this is the only way to obtain optimal results.

Read More

Answer

Many thanks for your valuable feedback on your recent experience of ab9484. In light of your feedback and that of several other customers we have removed all stock of this antibody and are offering our apologies for this unusual problem and a refund or replacement vial of another antibody. We have found that some customers are having difficulty with this product, while others are delighted with the same lot, we are very puzzled as to the reason for this and are investigating this extremely seriously in the laboratory. The quality of our antibodies is of utmost importance to us and we are very concerned about this case. We do not have an expected date for the new lot and therefore I would like to offer you a refund on your orders or three free vials of our other polyclonal anti GAPDH antibodies: https://www.abcam.com/index.html?datasheet=9483 https://www.abcam.com/index.html?datasheet=9485 I look forward to hearing from you regarding which you would prefer as well as your details of your orders to arrange this for you. Once again please accept our apologies for this unfortunate rare event, I hope this will not deter you from purchasing from Abcam in the future.

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Question
Answer

The only difference between these two antibodies is that ab9482 is the HRP-conjugated version of ab9484. The advantage of ab9482 being HRP-conjugated is that there is no need for a secondary antibody.

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Answer

Please take a look at the following publications: http://www.jci.org/cgi/content/full/112/7/1049 http://www.blackwell-synergy.com/links/doi/10.1046/j.1464-5491.2000.00231-2.x/abs/ http://www.jasn.org/cgi/content/full/10/9/1931 http://147.52.72.117/IJMM/2004/volume14/number1/55.pdf

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Question
Answer

Yes, you can use an anti-mouse IgG (H&L) secondary or a subclass specific secondary (to IgG2b), whichever you prefer

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

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