Anti-DOPA Decarboxylase antibody [DDC-109] (ab49916)

Thank you for your e-mail.
Please find here below our customer's reply:


As regards to the Abcam response, we have already verified that it is not a problem of secondary antibody (as you can well see in panel B of the first figure in which the primary Ab changes, but the secondary is the same, we also use the same secondary habitually with different primary Abs on lysates from CHO and we never found any problems). Nor it is a problem of overloading. In fact in panel B it was loaded the exact same amount of lysate as in that panel A. I also tried to dilute the Ab 1:10000 instead of 1:1000 and the situation does not change

We therefore ask a refund.

Thanks in advance

Kind regards

ANSWER:

Thank you for your response and for passing the customer's message.

It seems that the main point has not been commented in this recent e-mail. Chinese hamster ovary (CHO) is not a tested species therefore it is not listed on the product datasheet under cross-reactivity section. Unfortunately, we do not have any data regarding this species to share with the customer. Nevertheless, this antibody may recognize this species but we can’t support it

Therefore, I would recommend running a good and well-established positive control alongside with the samples. For this purpose as the datasheet indicates bovine or human or rabbit brain extract can be applied.

Has the customer run any of these samples as good control?

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

Let me know if you have any further questions.

Inquiry: Our PO is: 962 (29/03/2012) 1)Antibody storage conditions: -20°C it has been divided in aliquots to avoid repeat freez and taw 2)Description of the problem: the antibody recognize an aspecific band at the molecular weight of DDC 3)Sample: Fresh CHO cell lysate in PBS 4)Sample preparation:PBS + Roche complet mini inhibitor cocktail 5)Amount of protein loaded: 15μg cell lysate per lane 6)Electrophoresis/Gel conditions: Reducing gel at 10% acrylamide 7)Transfer and blocking conditions: nitrocellulose iBlot (Invitrogen) 7 minutes, Blocking in BSA 5% / milk 5%, 37°C for 1 h 8)Primary Antibody: we have tried the antibody at the manufacter diluition (1:1000) in BSA 5% and also at 1:10000 diuition. Antibody was incubated over night at 4°C and than we performed 3 wash in TBST buffer before the secondary antibody incubation 9)Secondary Antibody: Anti mouse HRP (GE Helthcare) diluition 1:2000 in BSA 5% , incubated 1h at room temperature and we performed 3 wash in TBST before the detection. We use this antibody from long time an it works very well. 10)Detection method: ECL at chemidoc (BIORAD) 11)Positive and negative controls used: We run only a negative control CHO cell not transfected . These cells do not have endogenous DDC so they don’t have to react with the antibody, but like you can see form the first lane in the picture this is not true. 12)How many times have you tried the Western?: 4 at different conditions 13)Have you run a "No Primary" control?: yes and we do not have any signal 14)Do you obtain the same results every time?: yes we always have an aspecific band at the weight of the DDC band 15)What steps have you altered?: none

ANSWER:

Thank you for your enquiry regarding ab49916 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Samples:

Chinese hamster ovary (CHO) is not a tested species therefore it is not listed on the product datasheet under cross-reactivity section. Unfortunately, we do not have any data regarding this species to share with the customer. Nevertheless, this antibody may recognize this species but we can’t support it

Therefore, I would recommend running a good and well-established positive control alongside with the samples. For this purpose as the datasheet indicates bovine or human or rabbit brain extract can be applied.

2) Loadingthe gel:

The left panel of the image you have kindly forwarded to us indicates that the gel may have been slightly overloaded. It seems that 15μg cell gave such a dense and strong signal. It may well be that the initial cell number used for sample preparation is very high and the non-specific signal may represent some degradation product. AT this stage it is rather difficult to identify the source of the problem.

Could you please specify how many cells were harvested in the RIPA buffer initially?

3) Secondary antibody only:

Has the customer run a no primary - only secondary antibody - control to see if any of the non-specific bands are due to the secondary or not? If you have not done yet, I would advise you to check it.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.