Anti-IDH2 antibody (ab55271)

In continuation to our previous correspondence, the customer tried your suggestions (she also diluted the antibody 1/1000 and usedNitrocellulose membrane). Unfortunately, these did not improve the results. The customer also performed WB with IDH2 antibody from Human Atlas (Sigma) and this showed the predicted product stronger than non specific, whereas Abcam's ab showed strong unspecific band and very weak or invisible specific bands. Please find the comparative gel image.

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.

Dear Tech Support Team,
Please find below the details of customer's complaint. Attached is the image of the gel.
Please advise.

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.

Bonjour, J'ai commandé un anticorps (anti IDH2, ref ab55271) mais rien n'est inscrit sur la fiche technique quant au blocage des sites non spé (avec serum bovine albumine ou avec du lait ? et à quel pourcentage ? ) Merci pour votre réponse Cordialement  

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.