Recombinant
RabMAb

Recombinant Anti-ACADM/MCAD antibody [EPR3708] (ab92461)

Reviews (3)Q&A (5)References (8)

Overview

  • Product name

    Anti-ACADM/MCAD antibody [EPR3708]
    See all ACADM/MCAD primary antibodies

  • Description

    Rabbit monoclonal [EPR3708] to ACADM/MCAD

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IP, IHC-P, ICC/IF, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human ACADM/MCAD aa 200-300. The exact sequence is proprietary.

  • Positive control

    • WB: Human heart or fetal liver tissue lysates; HeLa, HepG2, or K562 cell lysates; IHC-P: Human liver, mouse liver, and rat stomach tissues; ICC/IF: HeLa cells; IP: Mouse heart lysate; FC: HeLa cells.

  • General notes

    Previously labelled as ACADM. 

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab92461 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 1/10000 - 1/50000. Predicted molecular weight: 47 kDa.
IP 1/10 - 1/100.
IHC-P 1/600. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.
ICC/IF 1/50 - 1/250.
Flow Cyt 1/60.

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

    see all

  • 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

  • Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    All lanes : Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution (Purified)

    Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates
    Lane 2 : Human heart lysates
    Lane 3 : Mouse heart lysates
    Lane 4 : Rat heart lysates

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

    Predicted band size: 47 kDa
    Observed band size: 43 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat stomach tissue sections labeling ACADM/MCAD with purified ab92461 at 1/600 dilution (1.02 µg/ml). Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ACADM/MCAD with purified ab92461 at 1:50 dilution (10 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
  • Flow Cytometry - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Flow Cytometry - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ACADM/MCAD with purified ab92461 at 1/60 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
  • Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution (Purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 47 kDa
    Observed band size: 43 kDa why is the actual band size different from the predicted?

  • Immunoprecipitation - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunoprecipitation - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    ab92461 (purified) at 1/30 dilution (2 µg) immunoprecipitating ACADM/MCAD in Mouse heart lysate.
    Lane 1 (input): Mouse heart lysate 10 µg
    Lane 2 (+): ab92461 & Mouse heart lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab92461 in Mouse heart lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue sections labeling ACADM/MCAD with purified ab92461 at 1/600 dilution (1.02 µg/ml). Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue sections labeling ACADM/MCAD with purified ab92461 at 1/600 dilution (1.02 µg/ml). Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Western blot - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    All lanes : Anti-ACADM/MCAD antibody [EPR3708] (ab92461) at 1/10000 dilution (unpurified)

    Lane 1 : Human heart lysate
    Lane 2 : fetal liver lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : HepG2 cell lysate
    Lane 5 : K562 cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP conjugated Goat anti-Rabbit Ig at 1/2000 dilution

    Predicted band size: 47 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)

    ab92461 (unpurified), at 1/100 dilution, staining ACADM/MCAD in formalin-fixed, paraffin-embedded Human liver tissue by immunohistochemistry.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Immunocytochemistry/ Immunofluorescence - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)

    Immunofluorescent staining of ACADM/MCAD in HeLa cells using ab92461 (unpurified) at 1/100 dilution.

  • Flow Cytometry - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)
    Flow Cytometry - Anti-ACADM/MCAD antibody [EPR3708] (ab92461)

    Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ACADM/MCAD with unpurified ab92461 at 1/50 dilution(10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

References

This product has been referenced in:

  • Zheng F & Cai Y Concurrent exercise improves insulin resistance and nonalcoholic fatty liver disease by upregulating PPAR-? and genes involved in the beta-oxidation of fatty acids in ApoE-KO mice fed a high-fat diet. Lipids Health Dis 18:6 (2019). Read more (PubMed: 30611282) »
  • Liu Z  et al. N-terminal truncated peroxisome proliferator-activated receptor-? coactivator-1a alleviates phenylephrine-induced mitochondrial dysfunction and decreases lipid droplet accumulation in neonatal rat cardiomyocytes. Mol Med Rep 18:2142-2152 (2018). Read more (PubMed: 29901150) »
See all 8 Publications for this product

Publishing research using ab92461? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-8 of 8 Abreviews or Q&A

Western blot abreview for Anti-ACADM/MCAD antibody [EPR3708]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (human hepatocytes)
Gel Running Conditions
Reduced Denaturing
Loading amount
15 µg
Specification
human hepatocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Abcam user community

Verified customer

Submitted Dec 28 2017

Immunocytochemistry/ Immunofluorescence abreview for Anti-ACADM/MCAD antibody [EPR3708]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK293)
Specification
HEK293
Blocking step
Serum as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Fixative
Formaldehyde
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Abcam user community

Verified customer

Submitted Apr 03 2017

Western blot abreview for Anti-ACADM/MCAD antibody [EPR3708]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain)
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Loading amount
30 µg
Specification
Brain
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Abcam user community

Verified customer

Submitted May 27 2014

Question

I have isolated mitochondrial, cytosolic and nuclear fraction using standard protocol, also repeated the western blotting with ab92461. I am herewith attaching my result. Band is present through out the sample. Intensity of the band is comparatively higher in cytosolic fraction   My question is, if it is specific for ACADM (mitochondrial) why there is a band in cytosolic fraction. All the bands are below 45KDa, ie below expected range.     Details of Image from the Left,first  2 bands - cytoplasmic fraction                   next 3 bands - Nuclear fraction                   Last 3 bands - Mitochondrial fraction     Looking forward for your suggestion. Please help me save my time.  

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Answer

I appreciate the time that has been spent on these experiments and would be pleased to arrange, in compensation, a replacement of ab92461 with another anti-ACADM such as ab110296. Alternatively I can also arrange a credit note. I look forward to hearing from you with details of how you would like to proceed. Could you also please send me one of the following : the name of the distributor and the date of purchase? www.abcam.com/ab110296

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Question

Kindly solve the problem with ab92461 antibody and also help me to save my time   

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Answer

Thank you very much for your email. My colleague is in contact with the laboratory in order to try to find out what the problem might is. Indeed we have to investigate this issue further as the quality of our products is most important. We have had no reason so far to suspect any problems with this antibody. Can you confirm also that using no primary antibody - or an alternative rabbit antibody - the secondary antibody alone does not give any signal? Thank you very much for your cooperation. I would like to apologize for the long delays to resolve this issue. In order to allow you to continue with your research meanwhile, I can offer you an alternative antibody against ACADM. I would like to suggest the ab110296, Anti-ACADM antibody [ clone 3B7BH7], a mouse monoclonal antibody tested in WB and on mouse, rat, cow and human. The specificity of this antibody has also been validated with mass spectrometry. https://www.abcam.com/index.html?datasheet=110296 Please let me know whether you would like to accept the alternative antibody. I am looking forward to hear back from you.

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Question

I am herewith attaching images of Western blot of ACADM and Beta actin. Please compare these  Regards  

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Answer

Thank you for sending the image. We can’t make assumptions for the specificity of an antibody because of its same size band specificity of as of beta actin. Many protein have similar sizes, so experimental evidence is always good to prove the cross reactivity of any ab. I have following recommendations; - Use beta actin negative control lysate e.g. skeletal muscle myocyte and heart muscle myocytes are negative for beta actin so the lysates of these will be a good positive control. ab29330; https://www.abcam.com/Skeletal-Muscle-Human-Tissue-Lysate-adult-normal-tissue-ab29330.html http://www.proteinatlas.org/ENSG00000075624/normal - There is no sequence similarity between ACADM and beta actin so the antibody can’t cross reacts with beta actin. I would suggest using purified beta actin protein as a positive control. http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20111102-154040-0003-65312871-pg - ACADM is generally present in mitochondrial matrix. Prepare the mitochondrial lysates by getting rid of beta actin to see if the antibody was able to detect the ACADM or not. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. PS: Let me know if you are interested in using lysates ab29330; I can offer you 50% discount.

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Question

Subject - enquiry regarding ab92461 [CCE3243834] Thank you very much for your reply. I have repeated the procedure and developed band for ACADM and Beta actin using ECL. It is found that Anti-ACADM detected the Beta actin band. That is why it showed band at 42KDa position. Kindly recomment  solution for this problem.   I am herewith submitting answers for your valuable question  - Was the lysates denatured in loading buffer?    It was lysed in loading buffer containing 100mM tris buffer (PH 6.8) , 10% SDS, Glycerol and beta mercaptoethanol.   We normally recommend our customers using 1X SDS loading buffer with boiling at 100C for 5 minutes.   - Could you provide an image? Regarding the band below 42 kDa; is this the only band observed?   - Could you specify at what temperature the tissues were treated with RIPA buffer?   Lysed samples were incubated in ice for 30-45min   Also give a breif description e.g. how the lysates were prepared?   Tissue were homogenized and transfered to precooled vial. After 30-45 min of incubation , centrifuged at 12000g for 15 min. supernatant is collected.   - Does the band resolved properly in gel?   Electrophoresis were run for 2 hrs on 12% acrylamide gel.   - Finally could you provide the Abcam order number? The order number give in your email does not match any order record for this product.           929195 I will look forward to receiving your reply soon  

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Answer

Thank you for contacting us. The antibody is specific against ACADM protein only; ab92461 should not recognize the beta actin protein. Could you please explain why you think the antibody is detecting the beta actin protein? In regards to the expected band size of ACADM which is 47 kDa, that may run at different molecular weight due to various post translational modifications however we can’t say the ab92461is detecting beta actin instead of ACADM. I would suggest using beta actin negative lysates as a control lysates for experimental proof e.g. heart muscle myocytes are negative for beta actin. Looking froward to hearing from you. Have a good day!

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Question

LOT NUMBER GR 15062-5 ORDER NUMBER 2117 DESCRIPTION OF THE PROBLEM Non-specific band SAMPLE Total protein from rat heart tissue PRIMARY ANTIBODY Abcam Rabbut monoclonal to ACADM (ab92461), 1:10000 dilution at 5% BSA at TBS-T Buffer at 4Degree , overnight. followed by 3x10min wash in TBST. DETECTION METHOD ECL or DAB POSITIVE AND NEGATIVE CONTROLS USED wistar heart tissue lysate ANTIBODY STORAGE CONDITIONS Kept at - 20. Used for Western blotting at a concentration of 1:10000. SAMPLE PREPARATION tissue ground with RIPA buffer along with Protease inhibitor cocktail. Centrifuged at 12000g for 15 min, Denatured the sample in boiling water bath for 3 minutes AMOUNT OF PROTEIN LOADED 40 microgram ELECTROPHORESIS/GEL CONDITIONS SDS PAGE (12% acrylamide) TRANSFER AND BLOCKING CONDITIONS Towbins buffer is used for transfer, time one and half hour at cold condition. 5% BSA is used as blocking agent (time one hr) SECONDARY ANTIBODY sigma anti-rabbit antibody Catalogue no:A5441. 1:2000 dilution in TBST with 5% BSA. One hour incubation at 4 degree. followed by 3X10min wash in TBST HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES The band obtained is below the band of Beta actin (42KDa). the expected band position is 47Kda.ie, band developed at non specific site

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Answer

Thank you for your enquiry regarding ab92461 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful. To identify the source of the problem I would like to ask few questions; - Was the lysates denatured in loading buffer? We normally recommend our customers using 1X SDS loading buffer with boiling at 100C for 5 minutes. - Could you provide an image? Regarding the band below 42 kDa; is this the only band observed? - Could you specify at what temperature the tissues were treated with RIPA buffer? Also give a breif description e.g. how the lysates were prepared? - Does the band resolved properly in gel? - Finally could you provide the Abcam order number? The order number give in your email does not match any order record for this product. I will look forward to receiving your reply soon.

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