Overview

  • Product name
  • Description
    Mouse monoclonal to IDH2
  • Host species
    Mouse
  • Tested applications
    Suitable for: IHC-P, Flow Cyt, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Human, Recombinant fragment
  • Immunogen

    Recombinant fragment: HYREHQKGRP TSTNPIASIF AWTRGLEHRG KLDGNQDLIR FAQMLEKVCV ETVESGAMTK DLAGCIHGLS NVKLNEHFLN TTDFLDTIKS NLDRALGR, corresponding to amino acids 354-452 of Human IDH2

Properties

Applications

Our Abpromise guarantee covers the use of ab55271 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
IHC-P Use a concentration of 3 µg/ml.
Flow Cyt Use 1µg for 106 cells.

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

 

ICC/IF Use a concentration of 10 µg/ml.
WB Use a concentration of 1 - 5 µg/ml. This antibody has only been tested in WB against the recombinant fragment used as immunogen. We have no data on the detection of endogenous protein.

Target

  • Function
    Plays a role in intermediary metabolism and energy production. It may tightly associate or interact with the pyruvate dehydrogenase complex.
  • Involvement in disease
    D-2-hydroxyglutaric aciduria 2
    Glioma
    enetic variations are associated with cartilaginous tumors such as enchondroma or chondrosarcoma.
  • Sequence similarities
    Belongs to the isocitrate and isopropylmalate dehydrogenases family.
  • Post-translational
    modifications
    Acetylation at Lys-413 dramatically reduces catalytic activity. Deacetylated by SIRT3.
  • Cellular localization
    Mitochondrion.
  • Information by UniProt
  • Database links
  • Alternative names
    • D2HGA2 antibody
    • ICD-M antibody
    • IDH antibody
    • IDH2 antibody
    • IDHM antibody
    • IDHP_HUMAN antibody
    • IDP antibody
    • IDPM antibody
    • Isocitrate dehydrogenase [NADP], mitochondrial antibody
    • Isocitrate dehydrogenase 2 (NADP+), mitochondrial antibody
    • mNADP-IDH antibody
    • NADP(+)-specific ICDH antibody
    • Oxalosuccinate decarboxylase antibody
    see all

Images

  • Western blot against tagged recombinant protein immunogen using ab55271 IDH2 antibody at 1ug/ml. Predicted band size of immunogen is 37 kDa
  • IDH2 antibody (ab55271) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human colon.
  • ICC/IF image of ab55271 stained Mcf7 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 (ab55271, 10µg/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.

  • All lanes : Anti-IDH2 antibody (ab55271)

    Lane 1 : IDH2 in transfected 293T cell line
    Lane 2 : Non-transfected lysate


    The blocking agent used is 5% milk.
  • Overlay histogram showing MCF7 cells stained with ab55271 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab55271, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Intlekofer AM  et al. Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations. Nature 559:125-129 (2018). Read more (PubMed: 29950729) »
  • Intlekofer AM  et al. L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH. Nat Chem Biol 13:494-500 (2017). Read more (PubMed: 28263965) »
See all 20 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for your message and for providing this further information.
I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time your customer has spent on these experiments, and would like to offer a free of charge replacement or credit note in compensation (providing the product has been purchased in the last 180 days). In order to arrange this, I would appreciate if you could confirm the order number and date of purchase?
Thank you for your continued cooperation. I look forward to hearing from you with details of how your customer would like to proceed.

Read More

Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear the customer has had difficulty obtaining satisfactory results from this IDH2 antibody.

The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality.

I appreciate the time spent in the laboratory and understand the concerns. It is regrettable the results have not been successful. However, a band around 51 kDa seems to be present (as expected from the Isocitrate dehydrogenase according to SwissProt ID P48735), and therefore I would like to offer some suggestions to help optimise the results:

1) For mitochondrial proteins such as IDH2 a specific lysis buffer (RIPA buffer) or even fractionation of cell compartments may help to optimise the results. Please find details attached in our guide.

2) Loading less sample may help to reduce the background: Usually, 20 -30 ug protein per lane is sufficient.

3) The choice of membrane may give high background: Nitrocellulose membrane is considered to give less background than PVDF, so I would recommend trying it with nitrocellulose membranes.

4) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. Also, use one blocking reagent throughout the protocol, i.e. if blocked with BSA then also use it in the antibody dilution buffers.

4) You might reduce the concentration of the primary antibody even further (to 1 ug/ml or even less).

5) The secondary antibody may be binding non-specifically or reacting with the blocking reagent. I would suggest to run a secondary control without primary antibody to assess this issue.

6) If possible, you could run the gel a bit longer to better separate the higher molecular weight proteins. You then might be able to distinguish different post-translationally modified IDH2 forms.

We are happy to offer this technical support. If these suggestions have not yet been tried, I would appreciate if you can consider trying the suggestions. I hope you would agree that it would be preferable if we can get the experiment working and giving good results.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Merci de nous avoir contactés. L'anticorps anti-IDH2 ab55271 a récemment été testé sur un lysat de cellules tranfectées. Une bande de 51 kDa, correspondant à la IDH2, a été observée. L'agent bloquant utilisé est le lait à 5%. La fiche technique de l'anti-IDH2 ab55271 sera mise à jour très prochainement. J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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