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

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Overview

Product name

Anti-GAPDH antibody [mAbcam 9484] - Loading Control
See all GAPDH products (9) ...

Description

Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control

Specificity

This antibody shows a single clean band in Western blotting on cell lysates (rat and chinese hamster lysates also show another slightly lower band).

Tested applications

ICC/IF, WB, IHC-P, Flow Cytmore details

Cross reactivity

Reacts with

Mouse, Rat, Rabbit, Chicken, Cow, Dog, Human, Pig, Xenopus laevis, Chinese Hamster

Immunogen

Full length native protein from human erythrocytes.

Properties

Form

Liquid

Storage instructions

Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.

Storage buffer

Preservative: 0.01% Sodium Azide
Constituents: PBS, pH 7.4

Concentration

Concentration information loading...

Purity

IgG fraction

Clonality

Monoclonal

Clone number

mAbcam 9484

Myeloma

Sp2/0-Ag14

Isotype

IgG2b

Light chain type

kappa

  • Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) image (enlarge)

  • Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) image (enlarge)

  • Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) image (enlarge)

Applications

Show applications key

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.

  • ShowHide2 Images

    ICC/IF

     ICC/IF: Use a concentrat...Read more →

    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.)

  • ShowHide1 Image

    IHC-P

     IHC-P: Use a concentrati...Read more →

    IHC-P: Use a concentration of 5 µg/mlPerform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • ShowHide1 Image

    Flow Cyt

    Four stars (1 Abreview) Flow Cyt: Use at an assa...Read more →

    Flow Cyt: Use at an assay dependent concentration.

Target

Function

Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. Participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis. Nuclear functions are probably due to the nitrosylase activity that mediates cysteine S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2 and PRKDC (By similarity). Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate.

Pathway

Carbohydrate degradation; glycolysis; pyruvate from D-glyceraldehyde 3-phosphate: step 1/5.

Sequence similarities

Belongs to the glyceraldehyde-3-phosphate dehydrogenase family.

Post-translational
modifications

S-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus.
ISGylated.

Cellular localization

Cytoplasm > cytosol. Nucleus. Cytoplasm > perinuclear region. Membrane. Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions.

Target information above from: UniProt accessionP04406 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010).

Information by UniProt

Alternative names

  • Aging associated gene 9 protein antibody
  • G3P_HUMAN antibody
  • G3PD antibody
  • G3PD antibody
  • G3PDH antibody
  • GAPD antibody
  • GAPDH antibody
  • GAPDH antibody
  • Glyceraldehyde 3 phosphate dehydrogenase antibody
  • Glyceraldehyde 3 phosphate dehydrogenase liver antibody
  • Glyceraldehyde 3 phosphate dehydrogenase muscle antibody
  • Glyceraldehyde-3-phosphate dehydrogenase antibody
  • MGC88685 antibody
  • OCAS p38 component antibody
  • OCT1 coactivator in S phase 38 KD Component antibody
  • Peptidyl-cysteine S-nitrosylase GAPDH antibody
see all

Anti-GAPDH antibody [mAbcam 9484] - Loading Control images:

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

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

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 polyclonal Secondary Antibody to 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.

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

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

Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 0.5 µg/ml + HeLa cell lysate

Secondary
Goat polyclonal Secondary Antibody to 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

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

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

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.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

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.

  Immunocytochemistry/ Immunofluorescence-GAPDH antibody [mAbcam 9484] - Loading Control(ab9484)

Immunocytochemistry/ Immunofluorescence-GAPDH antibody [mAbcam 9484] - Loading Control(ab9484)

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.

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

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

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

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

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.

Image courtesy of Dr Mahesh Shivananjappa by Abreview.

See Abreview

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

This product has been referenced in:

  • Maddox SAet al. Early growth response gene 1 (Egr-1) is required for new and reactivated fear memories in the lateral amygdala. Learn Mem 18:24-38 (2011). WB; Rat.Read more (PubMed: 21177377) »
  • Wu Wet al. Changes in PGRMC1, a potential progesterone receptor, in human myometrium during pregnancy and labour at term and preterm. Mol Hum Reprod 17:233-42 (2011).Read more (PubMed: 21131300) »

See all 57 publications for this product

Publishing research using ab9484? Please let us know so that we can cite the reference in this datasheet

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