Anti-6X His tag® antibody (HRP) (ab1187)

Product code: 1187
Lot number: GR68013-1

Inquiry: Hello, I bought you anti 6X his antibody and have tried to use it on proteins that have a 10x his tag but poor signals in ELISAs. Do you know if it's binding efficiency is likely to be the same for 10xhis tagged proteins? Many thanks,

ANSWER:

Thank you for your inquiry.

I asked the lab if they tested this antibody with 10 X HIS tagged proteins. Unfortunately, they did not test this and therefore we do not know if the binding efficiency will be good.

I can suggest to use it on a protein with 6 x HIS tag as a positive control. If it binds well, it is not the antibody that proves to be the problem. If ab1187 does not deliver good results with 6 x HIS tagged proteins, then our Abpromise guarantee will apply.

I am sorry I could not be more helpful on this occasion and hope this information is nevertheless helpful.

Inquiry: Dear Sir/Madam, I am looking for a His-tag probe/antibody, which is already conjugated with HRP, for faster detection of His-tagged recombinant proteins. I would like to know if such a product exists in your product line, and if so, can I get a quotation for that product(s), and also, I would like to know the molecular weight for that product(s). Thank you. Regards,

ANSWER:

Thank you for your inquiry.

We have a number of antibodies in our catalog that might be of interest to you. I would like to direct you to the search facility on our website (www.abcam.com).

Please enter the name of the protein into the search box at the top of the home page. A list of products will be generated which can be further refined using the filters on the left hand side of the page. Using these filters will help you to identify products that have been tested in the applications and species you are interested in.

Here are a few antibodies which may be of interest to you:

- ab1187

- ab1269

- ab3553

- ab61209

I would advise you to take a look at the on-line datasheets for further information on availability/specificity/unit size/delivery etc

I hope this information is helpful to you. If you have specific questions about the products identified during the search, please do not hesitate to contact me.

Dear Sir/Madam,
We are trying to develop an ELISA for Dengue NS1 detection and since we have been been working for a long time with your products, we have decided to use your dengue products. We are in the process of validating our immunocapture system. The capture antibody we are using is a monoclonal antibody against DEN1-4 NS1 (ab41616) that we purchased together with theDengue NS1 glycoprotein (ab64456). We have already validated the presence of the mouse monoclonalantibody against DEN1-4 NS1 (ab41616) in our ELISA support using a Goat polyclonal Anti-mouse (ab20043). After looking at the citation for themouse monoclonalantibody against DEN1-4 NS1 we are expecting a good immunocapture. However on our initial tries, we cannot detect the presence of the dengue NS1 antigen. So we checked some details in the technical support and we found out that the NS1 antigen has a histidine tag which have been missing in the initial data sheet we had. And so we decided to purchase lasttime a unit of Anti-histidine tag HRP (ab1187) to check on theDengue NS1 glycoprotein (HisTag) (ab64456) but unfortunately even at 2 ug/ml concentration, we are not able to detect its presence. We are thinking of the possibility that the antigen we received may not have histidine tag as initially stated in the product sheet? but as you have guaranteed on our previous inquiry, the antigen is containing histidine tag. As loyal costumers of your company, we would like to ask you for the possibility of requesting replacement of the dengue NS1 glycoprotein (ab64456) that we have initially purchased with a different batch preferably to proceed with our experiments.
We are looking forward in working further with your dengue products.
Thank you for your usual costumer support.
Regards,

ANSWER:

Thank you for contacting us.
I am sorry to hear you are having problems with one of our products. I appreciate the details provided. I am afraid that product ab41616 is not guaranteed to work in ELISA. We have tested this antibody in WB and ICC, but we are not aware of its suitability for ELISA assays.
As far as I understand you are performing an indirect ELISA, coating your plate with the antigen ab64456 and then capturing it with the monoclonal antibody, using ab20043 (secondary) to detect your primary, is that correct? It could well be the primary antibody is not recognising ab64456 as it may not work for this application.
I can also confirm the peptide ab64456 is tagged with 6 histidines. It is also possible the antibody is not detecting this protein if the epitope it recognizes is masked with the histidine tags.
If you wish to I can try to find out the epitope targeted by ab41616 and check whether it is comprised within the tag region.
Regarding the failure of ab1187 (Anti-6X His tag® antibody (HRP)) to detect the glycoprotein, it could be due to the presence of sodium azide to preserve the protein. The antibody is linked to HRP and this enzyme is very sensitive to sodium azide.
In any case, as all of our products are covered by our Abpromise guarantee, if you wish to receive a replacement for ab64456, I will be more than happy to send it to you.
However, if you want to receive the same protein, you may still experience the same problems. In order to target this protein, I can try to find within our catalogue a primary against it that does not detect the epitope within the tagged sequence, or any other solution you may prefer.
I look forward to receiving your reply.

We tried to perform a competitive ELISA with an anti NS1 pAb coated on
the plate and with an anti His Tag (6x) pAb purchased from your company
(Prod # ab 1187). When we performed a control
experiment containing different dilutions of antigen NS1 WITHOUT His Tag
we got almost a linear response (see also attached data). The company
confirmed us twice that this competitor protein is NO His Tag fusion
protein. It would be very friendly if you had a suggestion why we got
such a high reading if there is no His Tag attached to the protein?
Thank you for your help.

ANSWER:

Thank you for contacting Abcam regarding ab1187.


I am sorry that you have been experiencing difficulties with this antibody in your ELISA experiments. Although the results resemble a somewhat linear response, I am not convinced the antibody is recognizing a specific target in your samples.


Have you tested different concentrations of antibody (both capture and detection)? How are you washing the plates? Have you tested a non-relevant protein?


Since there is such a high degree of variation among duplicates, I am not convinced that the HIS antibody is the main issue. Would you please provide additional details of your protocol?


I look forward to your reply so that I may assist you further. Please do not hesitate to contact us if you have any additional questions.

Actually, our vial is not amber. I suppose it should then be stored in an light proof secondary container?

We think we lost activity because it was not properly protected from light.

ANSWER:

Thank you for your reply.

This is very odd. I have asked our Goods-In department to check the color of the vials just to make sure that we store and deliver this product in the right container. I will get back to you whenI know more.

In the meanwhile, may I ask to fill out our questionnaire?The questionnaire for the Abpromise guarantee is an important tool for us as it represents a base for in-house investigations to target and resolve the problems that customers can experience as quickly as possible.

Thank you for your patience and cooperation.