Overview

  • Product name
    Anti-ACADM / MCAD antibody [3B7BH7]
    See all ACADM / MCAD primary antibodies
  • Description
    Mouse monoclonal [3B7BH7] to ACADM / MCAD
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IP, Flow Cyt, In-Cell ELISA, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Full length protein corresponding to ACADM/ MCAD.

  • Positive control
    • HeLa cells, HL-60 cells, Human cerebellum, HepG2 cells, HeLa cells, H9C2 (rat cells), and MEF (mouse cells) lysates.
  • General notes

    This antibody clone is manufactured by Abcam.

    MCAD (medium-chain acyl-CoA dehydrogenase) is an oxidoreductase enzyme of the mitochondrial fatty acid beta-oxidation pathway that is specific for acyl chain lengths of 4 to 16. It also utilizes the electron transfer flavoprotein (ETF) as electron acceptor that transfers the electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF dehydrogenase).

    Product was previously marketed under the MitoSciences sub-brand.

    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.

    Previously labelled as ACADM. 

Properties

Applications

Our Abpromise guarantee covers the use of ab110296 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 0.125 µg/ml. Predicted molecular weight: 47 kDa.
IP Use at an assay dependent concentration.
Flow Cyt Use a concentration of 1 µg/ml.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

In-Cell ELISA Use a concentration of 1 µg/ml. (0.1 µg/well)
IHC-P 1/1000. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 µg/ml.

Target

  • Function
    This enzyme is specific for acyl chain lengths of 4 to 16.
  • Pathway
    Lipid metabolism; mitochondrial fatty acid beta-oxidation.
  • Involvement in disease
    Defects in ACADM are the cause of acyl-CoA dehydrogenase medium-chain deficiency (ACADMD) [MIM:201450]. It is an autosomal recessive disease which causes fasting hypoglycemia, hepatic dysfunction, and encephalopathy, often resulting in death in infancy.
  • Sequence similarities
    Belongs to the acyl-CoA dehydrogenase family.
  • Cellular localization
    Mitochondrion matrix.
  • Information by UniProt
  • Database links
  • Alternative names
    • ACAD 1 antibody
    • ACAD1 antibody
    • Acadm antibody
    • ACADM_HUMAN antibody
    • Acyl coenzyme A dehydrogenase antibody
    • Acyl coenzyme A dehydrogenase C 4 to C 12 straight chain antibody
    • FLJ18227 antibody
    • FLJ93013 antibody
    • FLJ99884 antibody
    • MCAD antibody
    • MCADH antibody
    • Medium chain acyl CoA dehydrogenase antibody
    • Medium chain fatty acyl CoA dehydrogenase antibody
    • Medium chain specific acyl CoA dehydrogenase antibody
    • Medium chain specific acyl CoA dehydrogenase mitochondrial antibody
    • Medium-chain specific acyl-CoA dehydrogenase antibody
    • mitochondrial antibody
    see all

Images

  • All lanes : Anti-ACADM / MCAD antibody [3B7BH7] (ab110296) at 0.125 µg/ml

    Lane 1 : HepG2 cell lysates
    Lane 2 : HeLa cell lysates
    Lane 3 : H9C2 (rat cells) lysates
    Lane 4 : MEF (mouse cells) lysates

    Lysates/proteins at 15 µg per lane.

    Predicted band size: 47 kDa

  • Immunocytochemistry image of ab110296 stained Human HeLa cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with ab110296 (1 µg/ml) for 2 hours at room temperature or over night at 4°C. The secondary antibody was (green) Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. DAPI was used to stain the cell nuclei (blue). Target protein locates mainly in mitochondria.
  • ACADM / MCAD immunohistochemistry in Human cerebellum visualized with ab110296 at 1/1000. MCAD immunoactivity is most intense in neuronal cell bodies, most notably in the large Purkinje cells. Note the distinctive subcellular localization of MCAD immunoreactivity in the Purkinje cell bodies.

  • HL-60 cells were stained with 1 µg/mL ab110296 (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.
  • HL-60 cells were incubated at 37&degC for 24h with vehicle control (0 &microM) and different concentrations of fenofibrate (ab120832). Increased expression of ACADM / MCAD in HL-60 cells correlates with an increase in fenofibrate concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10&microg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab110296 at 1 &microg/ml and ab9484 at 1 &microg/ml overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP (ab97040) at 1/10000 dilution and visualised using ECL development solution.

     

References

This product has been referenced in:
  • Lim SC  et al. Loss of the Mitochondrial Fatty Acid ß-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function. Sci Rep 8:153 (2018). WB ; Human . Read more (PubMed: 29317722) »
  • Shaltouki A  et al. Alpha-synuclein delays mitophagy and targeting Miro rescues neuron loss in Parkinson's models. Acta Neuropathol 136:607-620 (2018). Read more (PubMed: 29923074) »
See all 7 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Heart tissue lysate)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
30 µg
Specification
Heart tissue lysate
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 30°C

Dr. SAIFUDEEN ISMAEL

Verified customer

Submitted Feb 15 2013

Answer

Vielen Dank für Ihre Antwort.

Leider ist mir bei meiner letzten Antwort ein Fehler unterlaufen und ich möchte diesen hiermit korrigieren:

ab110296 wird nicht im Kit ACADM ELISA Kit (ab129734) verwendet.. Es wird ein Antikörper verwendet, der nicht einzeln erhältlich ist. Bitte entschuldigen Sie vielmals diese Fehlinformation.

Bezüglich der anderen Fragen habe ich unsere Spezialisten bei MitoSciences gefragt. Ich habe ihnen die Antwort direkt unten rein kopiert:

"We purchased the cell line with the K304E (984A>G in ACADM gene) from the Coriell repository. The cell line is homozygous for 984A>G . The mutation in this cell line was found with a decrease in MCAD activity (12% from control) by the submitter, but there is no specific reference for this particular cell line in the coriell website.

http://ccr.coriell.org/Sections/Search/Sample_Detail.aspx?Ref=GM07844&PgId=166



However, it is well known in the literature that the K304E aa change (homozygous or compound heterozygous) induces MCAD deficiency (measured by enzyme activity). References below. Many of these studies were done before there were commercially available MCAD antibodies. So the WB data does not exist. Therefore we tested the above cell line from Coriell with our antibody 3B7BH7 and found that MCAD protein levels are significantly decreased (both by WB and by ICC). The customer can look at the data in the ICE booklet as mentioned in my previous email. We don’t have WB data with the antibody used in the kit showing control vs deficient, because the antibody is WB negative.

A good place to look at mutations known to induce MCAD deficiency is OMIM.

http://omim.org/entry/201450



http://omim.org/entry/201450#reference32 reported the findings in a prospective neonatal screening program in Pennsylvania using tandem mass spectrometry. From the first 80,371 newborns screened, they found 9 babies with MCAD (1/8930), plus 2 additional newborns, screened because of a previously known family history. Molecular analysis showed that 56% of the patients were compound heterozygotes for the 985A-G mutation (K304E; http://omim.org/entry/607008#0001), commonly referred to as G985, and a second mutation.


In 9 patients with MCAD deficiency, http://omim.org/entry/201450#reference16 found a 985A-G transition which resulted in a lys304-to-glu substitution (K304E; http://omim.org/entry/607008#0001) in the mature protein. These patients were unrelated, suggesting a high incidence of this abnormality among Caucasian patients. The change was not found in 20 healthy Caucasian and 6 healthy Japanese subjects. http://omim.org/entry/201450#reference16 found this point mutation in 31 of 34 (91%) mutant MCAD alleles. http://omim.org/entry/201450#reference31 estimated that 90% of MCAD cases involve the same mutation.

http://omim.org/entry/201450#reference33 characterized the molecular defect in 4 patients with mild MCAD deficiency. In routine neonatal screening on the fifth day of life, they had been found to have abnormal acylcarnitine profiles indicative of MCAD deficiency. Two were of German origin and the other 2 were born to different consanguineous Turkish parents. In all 4, the clinical course and routine laboratory investigations up to the age of 6 months were unremarkable. Enzyme studies showed residual MCAD activities between those with classic MCAD deficiency and heterozygotes. In 2 cases, ACADM gene analysis revealed compound heterozygosity for the common K304E mutation (http://omim.org/entry/607008#0001) and the 199T-C mutation (Y42H; http://omim.org/entry/607008#0011), which they designated Y67H. In the 2 children of consanguineous parents, homozygosity was found for the gly267-to-arg mutation (G267R; http://omim.org/entry/607008#0003) and the S220L mutation (http://omim.org/entry/607008#0012), respectively. As in other metabolic disorders, the distinction between 'normal' and 'disease' in MCAD deficiency is blurred into a spectrum of enzyme deficiency states caused by different mutations in the ACADM gene potentially influenced by factors affecting intracellular protein processing."



Ich hoffe, diese Information ist hilfreich und wird Ihnen Ihre Bedenken bezüglich des Kits nehmen.

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Answer

Vielen Dank für Ihre Anfrage.

Das Immunogen für ab110296 war das Protein voller Länge. Das Epitop, das der Antikörper bindet ist leider nicht näher kartiert worden und wir kennen es deshalb nicht.

Ich freue mich Ihnen bestätigen zu können, dass ab110296 im Kit ACADM ELISA Kit (ab129734) verwendet wird.

Es tut mir leid, dass ich Ihre Fragen diesmal nicht vollständig beantworten konnte und hoffe, dass diese Information dennoch hilfreich ist.

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